Innovative Food & Plants

Empowering Industry R&D: Uniform flesh quality for premium market positioning of blue swimmer crabs

Project No. 2007/244

R. Musgrove and S. Slattery

January 2009

Government of South Australia PP_090027 This publication may be cited as:

Musgrove, R. and Slattery, S. (2008) Empowering Industry R&D: Uniform flesh quality for premium market positioning of blue swimmer crabs. Final report to FRDC Project 2007/244. SARDI Publication No. F2008/000942-1. 90 pp.

This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission.

The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. It is a statutory authority within the portfolio of the federal Minister for Agriculture, Fisheries and Forestry, jointly funded by the Australian Government and the fishing industry.

SARDI Publication No. F2008/000942-1 Research Report Series No. 325 ISBN 978-1-921563-04-1

Authors: R. Musgrove and S. Slattery

Reviewers: Adrian Linnane & Andrew Barber

Approved by: Andrew Pointon

Signed:

Date: 9 December 2008 Distribution: Public domain Circulation: Fisheries Research and Development Corporation, Collaborators and Libraries

2 TABLE OF CONTENTS

NON TECHNICAL SUMMARY ...... 8 ACKNOWLEDGEMENTS ...... 10 1. BACKGROUND ...... 11 2. NEED...... 11 3. OBJECTIVES...... 12 4. METHODS...... 12 4.1 THE BLUE SWIMMER CRAB HANDLING CHAIN IN CARNARVON ...... 12 4.2. THE CRABS ...... 15 4.2.1 Data Collection ...... 15 4.2.2 Experiments...... 22 4.3 STATISTICAL ANALYSIS...... 24 5. RESULTS...... 25 5.1 ONBOARD HANDLING ...... 25 5.2 FACTORY PROCESSING ...... 27 5.2.1 The Precook Chiller...... 28 5.2.2 Cooking ...... 29 5.2.3 The Postcook Chiller ...... 34 5.2.4 Summary ...... 37 5.3 THE CRABS ...... 38 5.3.1 General...... 38 5.3.2 Cooking and Time of catch...... 38 5.3.3 Transport, cooking, and chilling...... 38 5.3.4 Cooking trials...... 47 5.3.5 Hepatopancreatic protease ...... 51 5.3.6 Mushiness ...... 54 5.3.7 Examination of the effect of snap freezing and liquid nitrogen storage on tissue levels of protease and peptide/free amino acids...... 58 5.3.8 Moult Stage/Shell Hardness ...... 59 5.3.9 Histology...... 60 5.3.10 Parasite load and Biochemistry ...... 64 6. DISCUSSION ...... 66 6.1 CRAB-RELATED ISSUES ...... 66 6.1.1 Moulting...... 66 6.1.2 Parasites ...... 69 6.1.3 Mitigation ...... 72 6.2 POST HARVEST HANDLING ...... 73 6.2.1 On-board handling...... 73 6.2.2 Green crab chiller ...... 73 6.2.3 Cooking ...... 77 6.2.4 Post Cook Chiller...... 78 7. BENEFITS AND ADOPTION ...... 81 8. FURTHER DEVELOPMENT ...... 81 9. PLANNED OUTCOMES...... 81 10. CONCLUSION ...... 83

3 REFERENCES ...... 85 APPENDIX 1: INTELLECTUAL PROPERTY ...... 88 APPENDIX 2: STAFF ...... 88 APPENDIX 3 BOX PLOTS...... 89 APPENDIX 4 CALIBRATION CURVE FOR PENETROMETER USED IN TISSUE QUALITY ANALYSIS...... 90

4 TABLE OF FIGURES

Fig 4.1 Seasonal fishing areas used by Abacus Fisheries, Shark Bay, Western Australia...... 13 Fig 4.2 Abacus Crab Factory (not to scale) ...... 14 Fig.4.3 Cooked crabs showing positions of cable ties used to close the incision and attach external logger...... 15 Fig 4.4 Sites used for testing shell hardness with the PTC lobster durometer...... 16 Fig 4.5 Use of a penetrometer for standard textural measurement of raw and cooked crab flesh...... 18 Fig 4.6 Live transport and cooling method experiements: flow chart ...... 23 Fig 5.1 Catching and initial cooling of crabs ...... 26 Fig 5.2 Processing of crabs at the factory ...... 27 Fig 5.3 Pre-cook chiller temperatures (ToC) over three days...... 28 Fig 5.4 Catch to factory crab core temperature profiles on the 4th and 6th of April 2008...... 29 Fig 5.5 Male crab core temperatures (ToC) during cooking on the 14/11 ...... 30 Fig 5.6 Time taken for crab core temperature to rise to 80oC during cooking (14- 19/11/07)...... 31 Fig 5.7 Time at or above 80oC for crab core temperatures during and immediately after cooking (14-19/11/07)...... 32 Fig 5.8 Maximum temperatures achieved during cooking (14-19/11/07)...... 33 Fig 5.9 Post cook chiller temperatures...... 34 Fig 5.10 Cooling of cooked blue swimmer crabs’ internal temperatures...... 36 Fig 5.11 Holding and cooking temperatures for female crabs caught on the 20- 21/11/07...... 37 Fig 5.12 Tissue firmness (TF) in crabs at each stage of the cold chain from boat to chiller/freezer...... 39 Fig 5.13 % Loss in weight of whole crabs after cooking by date/sex for live vs. iced and chilled vs slurry treatments ...... 40 Fig 5.14 % Weight loss on cooking for premoult, postmoult and mushy crabs in comparison to % weight loss data for that period (box plots)...... 42 Fig 5.15 Muscle polypeptides/ amino acids (μg/g wet weight) of crabs at each stage of the cold chain from boat to chiller/freezer...... 43 Fig 5.16 Muscle PI crabs at each stage of the cold chain from boat to chiller/freezer...... 44 Fig 5.17 Total muscle protein (% wet weight) in crabs at each stage of the cold chain from boat to chiller/freezer...... 45 Fig 5.18 Total muscle moisture (%) in crabs at each stage of the cold chain from boat to chiller/freezer...... 46 Fig 5.19 Percentage weight loss (+SE) in whole crabs during cooking trials on the 2- 3/04/08 and the 6-7/04/08...... 47

5 Fig 5.20 Cooking experiments: TF, % moisture, PI and PP_AA (+SE) for male crabs cooked at up to three temperatures (80, 90 and 100oC)...... 48 Fig 5.21 Crab core temperatures during cooking at 80, 90 and 100oC...... 50 Fig 5.22 Decline in hepatopancreatic protease activity (μg/g) with temperature...... 52 Fig 5.23 Biochemical profiles of crabs with mushy flesh (coloured symbols) compared with non-mushy crabs from the same day (box plots)...... 55 Fig 5.24 Effect of snap freezing and ice storage on hepatopancreas protease (μg/g) and muscle peptides/free amino acids (μg/g)...... 58 Fig 5.25 Relationship between TF in pre or postmoult (symbols) and intermoult (boxplots) crabs...... 59 Fig 5.26 Parasites found in blue swimmer crab muscle tissue...... 60 Fig 5.27 Parasite load and associated pathology: November 2007...... 62 Fig 5.28 Parasite load and associated pathology: April 2008 ...... 63 Fig 5.29 Proteolysis index and infection category...... 64 Fig 5.30 Parasite presence /absence and muscle protease activity (μg/g wet weight) ...... 65 Fig 6.1 Existing Abacus Crab Factory (not to scale)...... 75 Fig 6.2 Suggested modifications to the green crab chiller ...... 76 Fig 6.3 Suggested modifications to cooked crab chiller (not to scale) ...... 80

2007-244

EMPOWERING INDUSTRY R&D: UNIFORM FLESH QUALITY FOR PREMIUM MARKET POSITIONING OF BLUE SWIMMER CRABS

PRINCIPAL INVESTIGATOR: Dr R Musgrove

ADDRESS: SARDI Innovative Food and Plants SA Food Centre Regency Park SA 5010

OBJECTIVES:

1 Determine the principle sources of variation in flesh quality in Blue Swimmer Crabs in Shark Bay, Western Australia.

2 Develop and apply a postharvest and/or processing strategy designed to reduce variation in flesh quality

7 NON TECHNICAL SUMMARY

Abacus Fisheries of Carnarvon (WA) have developed new markets and products, yielding significant premiums and substantial increases in BSC beach price. However, the occurrence of mushy flesh can slow processing through excessive time spent checking for flesh quality. This impacts profitability and can threaten the super-premium fine dining market position enjoyed by Abacus Fisheries’ products.

This project clarified the causes of flesh deterioration and provided advice as to alternate harvesting and processing strategies.

Several issues have become apparent, particularly with regard to:

• The presence of parasites, and their impact on flesh quality. Two groups of parasites (Hematodinium spp and Acanthocephalans) were provisionally identified using histological methods with the former known to have significant impacts on flesh quality and mortality in other crab fisheries around the world.

• The impact of the moult cycle stage on post-cooking flesh quality. Postmoult crabs have reduced texture on cooking due to an increase in flesh moisture content.

• The likely impact on flesh quality of cooking time; time taken for crab core temperature to reach 80oC and post cook chilling.

A practical method is suggested for monitoring the impact of moulting periods. Postmoult crabs may be graded out using a simple squeeze test either on the boat sorting table or at the loading of the cooking conveyor at the factory. The former would be the most efficient and least wasteful. Soft crabs could also be returned to the factory for hardening if desired. The durometer used in this study could be utilised to standardise squeeze-test measurements.

A cooked flesh monitoring program is suggested to provide information on the periodicity and extent of mushy flesh (whatever the cause). The removal of mushy crabs from the processing line is of particular importance as poor quality meat from one crab would spread through the picked meat, having a broader impact than simple numbers might suggest. The penetrometer used to measure tissue penetration in this study could be utilised to standardise flesh quality between pickers, with a reading of less than 3 corresponding to “mushy”. The method is

8 obviously not available for those crabs (A-grade males and females) that are cooked, frozen whole and sent to market. Further parasitological studies are needed, with information required on variation in prevalence by area and season, and external signs which may be useful in grading before cooking. Previous research from other species should also be taken into account.

Suggestions are made for alterations to boat and factory infrastructure, particularly cooking and cooling processes. Onboard handling could be improved, particularly with regard to the present storage of crabs on deck once conventional storage bins are full. Reduced cooking time by pre-warming crabs and ice slurrying after cooking would improve cooking efficiency, reduce tissue breakdown and crab weight loss. In addition, limiting connectivity between the factory/outside and the chiller would reduce the chiller unit workload/running costs and maintain product quality.

KEYWORDS: Blue swimmer crab, Portunis pelagicus, flesh quality, moult stage, post harvest handling, cooking, chilling, temperature, parasite, Hematodinium, Acanthocephalan.

9 ACKNOWLEDGEMENTS

Thanks to Pete and Sandy Jecks for their kindness, help and enthusiasm. Thanks to Zack Jecks and the crew of the Joan J for supplying the crabs and for their patience with two extra bodies onboard. Thanks also to Abacus factory staff for allowing the authors to get in their way.

Thanks to Richard Stevens (WAFIC) and Dr Paul McShane (Global Marine Resource Management, Tasmania) for support and advice on the project. Many thanks also to Professor Brian Jones and Paul Hillier (Fish Health Laboratory, Department of Agriculture, Western Australia) for so much help at the WA end, including initial parasite identification. Thanks to Professor Jeffrey Shields (Virginia Institute of Marine Science, USA) and Dr. Sebastian Gornik, School of Botany, University of Melbourne) for advice, and parasite identification.

Finally thanks to FRDC TRF program for supplying funds for the project.

1. BACKGROUND

The Shark Bay blue swimmer crab (BSC, Portunis pelagicus) fishery is the largest single blue crab fishery in Australia. In 2004/05 the catch was 726t (Bellchambers, et al., 2006), eight times higher than any other area in WA and approximately the same as the catch from the entire Queensland coast (690 - 722t, 2004/05 - 05/06, QDPIF, 2007). The catch is also greater than that in South Australia (2004/05 – 614t, Currie and Hooper, 2006) and much higher than New South Wales (160 – 175t, 2004/05 - 2005/06, NSWDPI, 2007) Approximately 600t per annum is taken by Carnarvon- based Abacus Fisheries, a vertically integrated company (i.e. involved with harvesting, processing and marketing). After processing (cooking and blast freezing) the product is sent to domestic Australian and export markets such as Japan, Europe and USA. Abacus Fisheries’ blue swimmer crab harvest returns $1.8-2.5M/ year to the company.

Post-harvest flesh quality is a significant issue in the fishery with a reported 10-20% of crab flesh unsaleable (i.e. mushy) after cooking. This project addresses the following objectives:

1 Determine the principle sources of variation in flesh quality in Blue Swimmer Crabs in Shark Bay, Western Australia.

2 Develop and apply a postharvest and or processing strategy designed to reduce variation in flesh quality

Two lines of enquiry were explored: 1) The impact of post harvest handling and processing (onboard and at factory) and 2) The impact of parasite species and load. The latter was included as some parasite species (i.e. Ameson spp, Hematodinium spp) have a significant impact on crustacean fisheries in other parts of the world (e.g. Alaskan tanner crab (Chionectes opilio, Shields, et al., 2005) and Norway lobster (Nephrops norvegicus) (Stentiford and Shields, 2005)

2. NEED

As stated above, BSC post-harvest flesh quality in Shark Bay WA is a concern to Abacus Fisheries. Mushiness in BSC may be caused by proteolytic enzymes escaping from the hepatopancreas post-mortem and breaking down adjacent muscle tissue (Slattery et al 1989). This can occur if crabs die before cooking (Stevens, 1995) or cooking times are too short or temperatures too low. Raising crab core

11 temperatures to greater than 80oC for 2 minutes may reduce the problem (Slattery, et al., 1989).

Notwithstanding the above, the problem persists at Abacus Fisheries (Shark Bay) despite cooking chilled crabs for 13 minutes at 95-97oC. This suggests further information is needed to resolve the issue. One possibility is that flesh quality is related to the moult stage of the crab. Muscle water content is highest in postmoult/early intermoult crustaceans (Musgrove, 2001), possibly contributing to mushiness on processing. Shark Bay crabs moult throughout the year although peaks appear around January/February, May/June and July/August (P. Jecks. pers. obs.).

Parasite infestation (i.e. microsporidians especially Ameson sp.) in harvested crabs may also result in mushy flesh after cooking (Shields and Overstreet, 2007). Stress may also affect tissue quality, influenced by predators (eg. octopus), harvest process or sub-optimum post-harvest holding, including chilling. The effect of gut-fullness is also unknown; fuller guts may generate greater protease activity, leading to more postmortem tissue breakdown.

Abacus Fisheries has developed new markets and products, yielding significant premiums and substantial increases in BSC beach price. However, the occurrence of mushy flesh can slow processing through excessive time spent checking for flesh quality to grade out affected product. This impacts upon profitability and therefore highlights the need to clarify the causes of flesh deterioration so alternate harvesting or processing strategies can be implemented.

3. OBJECTIVES

1 Determine the principle sources of variation in flesh quality in Blue Swimmer Crabs in Shark Bay, Western Australia.

2 Develop and apply a postharvest and or processing strategy designed to reduce variation in flesh quality

4. METHODS

4.1 The blue swimmer crab handling chain in Carnarvon

Two field trips were undertaken to Abacus Crab Pty Ltd processing facility in Carnarvon, Western Australia. The first took place from the November 12th - 22nd,

12 2007, the second from March 31st - April 10th, 2008. In November crabs were sampled on the western side of Shark Bay; in April samples were taken on the eastern side, 30km south of Carnarvon. Areas sampled were defined by commercial fishing operations (Fig 4.1).

Fig 4.1 Seasonal fishing areas used by Abacus Fisheries, Shark Bay, Western Australia

Carnarvon

Shark Bay

Fishing Areas November to February March to June 50 km

Perth Sydney Adelaide

13 The postharvest handling chain was studied by the authors and photos were taken at significant points along the chain. Air, water, and internal crab temperatures (and time of sampling) were logged using iButton data loggers (Dallas Maxim). The chain was followed from the initial on-board storage of blue swimmer crabs on ice or in live tanks through to the green crab chiller at the factory (Fig 4.2), the cooker and finally the blast freezer or cooked crab chiller.

Fig 4.2 Abacus Crab Factory (not to scale)

FreezersStore

Blast Green Crab Freezer Freezer (-22oC) Chiller (-28oC)

A Cooked Crab Chiller Packer

Tea Packer room B

Wash room / WC Office

Weighing station Store

C Cooker D Shrink wrapper

A = Green crabs loaded B = Damaged and undersized crabs removed C = Crabs transferred into cooker from conveyer via tipping bucket Picking Store Store Room D = Cooked crabs into hopper then down to manual sorting station Conveyor

Green crabs

Cooked crabs

14 4.2. The Crabs

4.2.1 Data Collection

Crab dissections were carried out and tissue samples taken on board, upon arrival at the factory, after cooking and the following day after chilling (females) or freezing (males) overnight. The sexes were treated differently as per industry practice. Manipulations were carried out to investigate the effect of live transport and rapid post-cook chilling on tissue quality. In each case, experimental baseline data were provided by dissection of 5-10 uncooked crabs on the boat and/or at the factory as appropriate.

4.2.1.1 Core Temperature

Crab core temperature was taken using a 3mm microprobe connected to a TPS WP80 pH conductivity temperature meter. Crab carapace width was measured with vernier callipers (to within 0.1mm, spine to spine as per WA Fisheries practice).

o High temperature data loggers (iButtonTM, Range: 0-125 C) were also inserted into 2 crabs on each boat sampling trip as follows. A horizontal incision was made just above the mandibles to allow the insertion of the data logger which was inserted deep into the body cavity using tweezers. As this procedure may have influenced tissue quality through disruption of the stomach and hepatopancreas, crabs with internal loggers were not used in futher analysis. Two long cable ties were then fastened around the crab and over the mandibles to effectively close the incision (Fig 4.3). One of the cable ties was also used to attach a second logger to the outside of the crab. Loggers were sealed into individual purpose-made plastic bags (Venus VHB-00 bag sealer) for protection. Crabs used for these loggers had been on ice for 30 minutes and were torpid.

Fig.4.3 Cooked crabs showing positions of cable ties used to close the incision and attach external logger.

Cable ties

iButton Logger sealed in plastic

15 4.2.1.2 Shell hardness

Shell hardness was assessed using a lobster durometer (Fig 4.4, PTC Instruments, USA) on selected sites. These were the relatively flat, smooth areas on the dorsal posterio-lateral margin of the carapace and on the ventral posterior sternal plate. Each site (left and right for each of dorsal and ventral surfaces) was tested only once per animal to avoid damaging the shell and influencing subsequent readings. Readings were consistent left to right on each surface.

Fig 4.4 Sites used for testing shell hardness with the PTC lobster durometer

4.2.1.3 Tagging

All crabs used in the trials, and not immediately dissected, were tagged as follows. Each crab had a coloured cable tie placed around a claw and a T-bar tag (Hallprint) put through the inside lateral margin of the third abdominal somite. The latter was selected as it is not well perfused with haemolymph and is as far away from the body musculature as possible. All tags survived the transport and cooking process.

4.2.1.4 Dissection.

The crab’s carapace was removed (i.e. the crab was “backed”), gut fullness was estimated (rated <25, 25-50, 50-75 or 75-100%). Muscle and hepatopancreas samples were then taken (2g/sample) and stored in 2ml cryotubes in liquid nitrogen for later biochemical analysis to determine proteolysis level and protease activity respectively (see below). Muscle samples were cut from the right paddle muscle

16 mass. Additional tissue samples were taken from the left paddle muscle mass for histological analysis. A paddle was also removed and put on ice for later moult stage assessment.

4.2.1.5 Texture and tissue firmness measurement

Muscle samples from raw and cooked crabs were subjected to oral texture and mechanical tissue firmness (TF) measurements during live transport/cooling method and cooking trials. TF measurement was carried out using a penetrometer with a shearing blade designed by Steve Slattery (Innovative Food Technology, QDPI) (Fig 4.5). Oral testing alone was carried out for the live transport and cooling methods experiment as the penetrometer was not available. Tested samples were from the swimming leg muscle block used for the biochemical analysis (refer Section 4.2.1.6).

17 Fig 4.5 Use of a penetrometer for standard textural measurement of raw and cooked crab flesh.

Designed by S Slattery, Innovative Food Technology, QDPI. a) Flesh (8-10g) was weighed to the nearest 1g using the 2 internal sleeves of the penetrometer and a plastic petri dish. There was no effect of tissue weight on the results over this range (P>0.05).

b) The remainder of the penetrometer (gauge plus sleeve housing) fits over the sleeves. The blades and how they fit into the sleeves are shown without the housing.

c) The penetrometer was placed over the sleeves and depressed with one motion, giving a measurement between 1 and 100. The penetrometer measurement is unit- less with a scale ranging from 0-100. The measurement is divided by the weight of the sample tissue to standardise readings within and between crabs. The calibration curve for the penetrometer is shown in Appendix 4.

There were no significant differences between TF measurements taken on the left and right sides of crabs (P>0.05) at any point in the postharvest chain so a median was derived from each pair of measurements and used in subsequent analysis.

4.2.1.6 Biochemical analysis

On arrival at the laboratory, all samples were transferred from liquid nitrogen to a -80oC freezer until analysed. Raw and cooked crab tissue was analysed as follows.

Hepatopancreatic and muscle protease activity was assayed fluorometrically (Hitachi F2000 Fluorescence Spectrophotometer) using a protease fluorescent detection kit (Sigma-Aldrich PF0100). A 1g sample of tissue was used. After initial thawing on ice, samples were weighed into tared tubes (to 0.001g) and equal v/w of chilled distilled water added. Samples were then homogenised on ice for 15-30 s and 1ml transferred to eppendorf tubes using a transfer pipette. Samples were then spun for 10 min at 15,000g and the supernatant transferred to 5ml screw-top tubes and frozen (-80oC) for later analysis using the above kit. The incubation buffer used in the analysis was 20mM sodium phosphate with 150 mM sodium chloride (pH 7.6), the assay buffer was 500mM Tris buffer (pH 8.5). Trypsin was used as the protease control and fluorescein isothiocyanate-labelled casein as the substrate.

Soluble muscle proteins (peptides and amino acids, PP_AA) were assayed spectrophotometrically using the Folin-Ciocalteu method (Micro Lowry Total Protein Kit Sigma TP0300/ L3540) in conjunction with a tyrosine standard curve; and total N with an elemental analyser (Elementar). Samples were homogenised as described above, then 2ml of 24% TCA added and the samples vortexed for 10 s. After standing on ice for 20 mins they were spun at 5,000g for 20 mins. The supernatant was transferred to 5ml screw-top tubes and frozen (-80oC) for later analysis using the above kit.

The degree of proteolysis (Proteolysis Index, PI) was calculated as the ratio of soluble protein to %total protein (as total N x 6.25) in the muscle.

4.2.1.7 Histological analysis

Histology was carried out on muscle and hepatopancreas tissue samples preserved in 10% neutral buffered formalin at a volume ratio of 1 part tissue to 10 parts formalin. Fixed tissues were embedded in wax, sectioned at 5 microns and stained with haematoxylon and eosin using standard procedures. Additional samples were preserved in 100% ethanol for PCR (polymerase chain reaction) analysis to independently confirm the identity of the parasites.

20 4.2.1.8 Examination of the effect of snap freezing and liquid nitrogen storage on tissue levels of protease and peptide/free amino acids.

The following procedure was undertaken to assess the effect of snap-freezing and liquid nitrogen storage of tissue samples on the biochemical parameters measured during the project. This was considered important for comparison of results with those of other studies were fresh sampes were taken.

Twenty live BSC’s were obtained from a local (Adelaide) fisherman and taken back to the laboratory in 90L insulated bins on ice. Crabs were then backed and cut in half sagitally and three sets of samples taken. Fresh hepatopancreatic and muscle tissue was removed from one half of each crab and either put straight into distilled water for homogenisation and analysis of protease or peptide/free amino acids as described earlier, or placed in 2ml cryotubes, and snap-frozen and stored in liquid nitrogen. The remainder of each crab was bagged, labelled and put on ice (in an insulated foam box) at 4oC and sampling of fresh and snap-frozen tissue for biochemical analysis repeated 3 days later.

21 4.2.2 Experiments.

Three sets of experiment were run, the first two (time between catch and processing, live transport/cooling method) set up from the point of harvest and the third (cooking trials) confined to the factory.

4.2.2.1 The effect of time from harvest to processing on tissue quality

Ten male crabs from each of the first and last strings of pots hauled on one day were tagged (refer Section 4.2.1.3) and placed in onion bags on ice. Temperature data o o loggers (iButtonTM, Dallas Maxim, Range: -40 C to + 40 C) were included in the o onion bags. High temperature data loggers (iButtonTM, Range: 0-125 C) were inserted in two further male crabs (Section 4.2.1.1) and these crabs also placed in the onion bags.

Gut temperatures were taken from five of each group (i.e. first and last pot lifts) of the above crabs upon arrival at the factory. They were then measured, and dissected with tissue samples taken (Section 4.2.1.4). The remaining five crabs, plus the two containing temperature loggers, were then put through the cooker. After cooling for 2 hours in the chiller, crabs were dissected and muscle texture assessed orally and visually (Slattery, 1989) (Section 4.2.1.5). Further samples were also taken for biochemical analysis (Section 4.2.1.6).

4.2.2.2 Live transport and cooling method

Thirty freshly caught crabs were tagged and split into two groups, 15 placed in crates in live tanks and 15 in onion bags on ice (Fig 4.6). The live tanks were flow through and thus at ambient seawater temperature (~22oC). External and internal temperature data loggers were included in both treatments.

Once at the factory, all 30 crabs were placed on ice in the green crab chiller. Five crabs from each treatment were then measured and dissected as described above and the remaining 10/treatment (plus crabs with high temperature loggers) sent through the cooker.

Once out of the cooker, equal numbers of male crabs (i.e. 5/treatment) were placed directly in the cooked crab chiller in air or in ice slurry, for two hours, then blast frozen at -28oC overnight. Females were subjected to the same chiller or slurry (2hr) treatments then left in the chiller overnight (5/treatment). The following day all crabs were dissected. In each case crabs were left in the chiller until dissected, gut

22 temperatures then taken immediately and tissue samples removed and stored in liquid nitrogen as described earlier.

The experiment was repeated four times, twice using male and twice using female crabs.

Fig 4.6 Live transport and cooling method experiements: flow chart

Boat Crabs Iced crabs Live crabs in onion dissected in insulated bags in on-board (5/treatment) bins tanks

Factory All into Green Crab Chiller (ref Fig 4.2)

Crabs Crabs cooked dissected (10/treatment) (5/treatment)

Females Males

Ice Slurry Chiller Ice Slurry Chiller

Chiller Blast Freezer

All remaining crabs dissected

23 4.2.2.3 Cooking trials

Two cooking trials were undertaken, on separate occasions, with the objective to assess the effect of cooking temperature on flesh quality and biochemical parameters. For each trial male crabs were selected from the catch upon arrival at the factory.

For the first trial ten crabs were measured, tagged then put into water at each of three temperatures (80oC, 90oC and 100oC) for 13 minutes, the time used for commercial cooking. Afterwards all crabs were put into ice slurry for 2 hours then transferred to the cooked crab chiller. Two additional crabs were included, containing high temperature data loggers.

The following morning (approximately 14 hours later) crabs were dissected and tissue samples taken for analysis as described earlier.

The second trial was similar but used two temperatures (80oC and 90oC) only.

4.3 Statistical Analysis

Data were analysed using SPSS 16.0 for Windows. GLM, Means, Regression and Nonparametric modules were used where appropriate. Significant results were accepted at P< 0.05. Box plots were used to illustrate the effects of mushiness and moult stage on biochemical parameters, and cooking on whole weight (Figs 5.15, 5.24, 5.25). Refer to Appendix 3 for an explanation of these plots.

24 5. RESULTS

5.1 Onboard Handling

The handling process is as follows. Crabs are caught using 450 baited crab pots, set in 9 lines of 50 pots. Pots are pulled line by line, between about 0600h and 1200h. Actual times will vary depending on distance to fishing grounds and catch on the day. Once each line is completed (i.e. all pots are on deck) it is rebaited and reset. Pot soak time is approximately 24hr.

Once on board, crabs are shaken, or manually removed, from pots into an ice slurry bin then sorted by sex and damage. They are then put into plastic crates and covered in ice, and the crates packed into large onboard insulated bins (Fig 5.1). Once the catch exceeds the capacity of the storage bins crabs are stored in crates either in non-insulated holds set into the deck or on the deck itself.

Crabs may be on ice for 1.5 to 10 hours depending on distance from port and time of catch.

25 Fig 5.1 Catching and initial cooling of crabs

Setting

Hauling

Crab removal from pot

Ice slurry bin

Storage

26 5.2 Factory processing

Upon arrival at the factory the crates are unloaded into a chiller (hereafter called “precook chiller”). From there they are individually placed on a conveyor system for cooking and other processing (Fig 5.2).

Fig 5.2 Processing of crabs at the factory

Loading the Cooking conveyer

A grade males/ females are Remaining B-grade and graded & shrink-wrapped damaged crabs go to chiller overnight and are picked the following morning.

picking

weighing

packing blast-freezing

27 5.2.1 The Precook Chiller

Temperatures in the precook chiller were monitored during the April trip. The precook chillers showed wide temperature variation, with the associated opening and closing of the chiller’s external sliding door corresponding to arrival of crabs from the jetty and their placement in the chiller (Fig 5.3). Loggers were set at the bottom of wall in the corner as far from the doors as possible. Crabs were in ice at this point and did not appear to be affected by these wide fluctuations, as indicated by internal loggers (Fig 5.4).

Fig 5.3 Pre-cook chiller temperatures (ToC) over three days.

Arrival of crabs at the factory

T0C 8

0 16 20 4 8 12 16 20 4 8 12 16 20 4 8 12 16 20 4 8 12 16 20 4 8 5/4/08 6/4/08 7/4/08 8/4/08 9/4/08 10/4/08 Date and time (24hr clock)

28 Fig 5.4 Catch to factory crab core temperature profiles on the 4th and 6th of April 2008. Loggers removed at beginning of the cooking process.

30 6/4/08 female 4/4/08 male 25 Internal temperature profile Insertion of loggers on boat

20 Into precook chiller

o Into cooker T C 15

0 6:00 8:00 10:00 12:0 Time (24 hour clock)

5.2.2 Cooking

Crabs are brought out in batches (10 or more crates) from the chiller and ice removed as they are loaded onto the conveyer. The cooker is set at 95-97C and crabs go through on a conveyer in 13 minutes. The day’s catch goes through to the cooker in approximately 4 hours with males sent through first, followed by females and damaged crabs.

Crab internal temperatures took most of the cooking time (i.e. 13 minutes) to get above 80oC (Fig 5.5), the recommended minimum cooking temperature (Slattery et al, 1989). For example, the outside temperature of crab C1 rose to 93oC immediately upon entry to the cooker (set to 95oC) (Fig 5.5). The internal temperature of the same crab took 11 minutes to get above 80oC; then stayed for 2 minutes at a maximum temperature of 82oC.

29 There was a large variation in time taken for internal crab temperature to reach 80oC (Fig 5.6), and in time spent at or above 80oC both within and between cooking days (Fig 5.7). Generally females appear to take the longest to reach the recommended 80oC and consequently spend the shortest time above that level. The cooker was set at 95oC for the early sample on the 14th of November, for the remainder of the project (Including April) the cooker was set at 97oC. This temperature change was a direct consequence of the discussions with the Abacus Fisheries based on data downloaded at the end of the experiments on the morning of the 14th of November.

Fig 5.5 Male crab core temperatures (ToC) during cooking on the 14/11 Crabs caught/processed early (C1-C9, left) and late (C12-C20) on the same day. One cooker temperature trace included; the logger was attached to the outside of C1. C = cooked crab. Fig 1 Core temperatures during cooking (14/11) for male crabs ca ught/processed early (C1 – C9, left) and late (C12 – C20) on the 14/11/07. One cooker temperature trace included; the logger was attached to the outside of C1. C = cooked crab. 100

80 C1 C12 C2 C13 70 C3 C14

C4 C16 60 C5 C18 C9 C19 ToC 50 Outside C1 C20 Target crab core 40 cooking temperature

0 12:20 13:00 13:40 14:20 15:00 15:40 16:20 17:00 Time (24 hr clock)

Fig 5.6 Time taken for crab core temperature to rise to 80oC during cooking (14- 19/11/07)

Average ( ) and range (vertical bars). F = female (pooled over dates); remaining data for males only. E = early (first pot line), L = late (last pot line). Minimum of three temperature readings per point.

8 Time (mins) 6

ELEL 16/11 19/11 F

14/11 15/11

Date (males)

Fig 5.7 Time at or above 80oC for crab core temperatures during and immediately after cooking (14-19/11/07)

8 Time (mins) 6

0 E LEL 16/11 19/11 F

14/11 15/11

Date (males)

32 Average maximum crab core temperatures reached were mostly around the 85 to 87oC mark (Fig 5.8) but again varied widely. Females recorded the lowest average maximum temperatures.

Fig 5.8 Maximum temperatures achieved during cooking (14-19/11/07).

Average ( ) and range (vertical bars). F = female (pooled over dates); remaining data for males only. Minimum of three temperature readings per point. E =

early (first pot line), L = late (last pot line). Target crab core cooking temperature .

100

T oC 85

70 E LEL 16/11 19/11 F

14/11 15/11

Date (males)

Cooked A-grade male and female crabs are shrink-wrapped then blast frozen (-25oC). B grade crabs and those with missing limbs or other damage are placed in the post-cook chiller overnight then picked for meat which is blast frozen in tubs

33 5.2.3 The Postcook Chiller

The postcook chiller showed significant temperature variations (Fig 5.9). The movement of batches of hot, freshly-cooked crabs from the factory into the chiller throughout each afternoon would significantly contribute to the periodical increases in temperature. Occasional opening of either the outside or the factory-side door would also contribute to the observed pattern.

Fig 5.9 Post cook chiller temperatures.

top of wall bottom of wall Placement of crabs in the chiller

8 0C 6

0 8 12 16 20 4 8 12 16 20 4208 12 16 41216208 41216208 48 5/4/08 6/4/08 7/4/08 8/4/08 9/4/08 10/4/08 -2 Date and time (24hr clock)

-4

These fluctuations undoubtedly influence the crab cooling process, with cooling times to 0oC ranging from 4h 28mins to 13h 37mins. In one case (6/4/08), the temperature took 13h 48mins to reach a minimum of 5oC and zero was not reached. The crab logged from 14:00h on the 8th of April (blue line) took 13h 37min to cool to 0oC whereas the crab logged on the same date but beginning at 20:00h, dropped more rapidly and took only 6 hours to get to 0oC (Fig 5.10). The crab from later in the day may have benefited from reduced activity in the chiller and gradual cooling of its load of crabs, most of which had been loaded at least two hours before. The chiller temperature also dropped below 0oC at about 02:00h which would have undoubtedly facilitated the cooling process.

34 These logged crabs were placed in two layers in crates adjacent to crabs destined for the picking room. The small number of crabs per crate probably accounts for the core temperatures tracking external (i.e. chiller) temperatures closely (Fig 5.10, 20/11/07). Full crates of crabs to be processed would have cooled more slowly because of the greater thermal mass.

Fig 5.10 Cooling of cooked blue swimmer crabs’ internal temperatures.

Time of day vs temperature (oC) and time (hours) to cool from maximum cooking temperature to minimum in chiller. Loggers were also mounted on the outside of crabs, one such trace ( ) is included for comparison.

100

60 Time to minimum temperature

(hours) o T C 50 19/11/07 4:45

19/11/07 4:28 40 20/11/07 7:52 20/11/07 7:54 30 6/04/08 13:48 8/04/08 13:37 20 8/04/08 6:21 10

0 14:00 16:00 18:00 20:00 22:00 0:00 2:00 4:00

Time of Day (24 hr clock)

36 5.2.4 Summary

In a typical example of the handling chain (Fig 5.11), crabs (in this case females) caught between 0600 and 0700 came off ice at (1-3oC) 6-7 h later and were put through the cooker and 10 minutes later reached a maximum core temperature of 81oC, remaining above 80oC for 2 minutes (maximum cooker temperature 93oC: pink line, Fig 5.11 ). Crabs (dark green line, Fig 5.12) then took 7-8h to reach 0oC in the chiller. Temperatures recorded for males and females were similar on the boat but typically males reached a core temperature of 87oC after approximately 8 mins in the cooker and remained above 80oC for 5 minutes. Cooling to 0oC in the blast freezer took about three hours.

Fig 5.11 Holding and cooking temperatures for female crabs caught on the 20- 21/11/07

100

80 Early females outside temperature

70 Early females core temperature

Late females outside temperature 60 Crab core temperature coming out of chiller (on ice) before cooking Temperature o ( C) 50 Target crab core cooking temperature Crab core temperatures coming out of cooker

40 Crab core temperatures between 18:04h and 18:45h (had been in chiller since 14:54h)

0 13:20 14:40 16:00 17:20 18:40 20:00 21:20 22:40 0:00 1:20 2:40 4:00 5:20 6:40 8:00 9:20 10:40

20/11 21/11

Time (24 hr clock) /Date

37 5.3 The Crabs

5.3.1 General

Crab size and harvest date had no significant effect (ANOVA P>0.05) on any of the following parameters. Harvest date was analysed in this context due to possible seasonal differences in crab condition impacting on biochemical parameters and flesh quality.

5.3.2 Cooking and Time of catch

Time of catch (i.e. first or last string of pots) and sex did not affect raw or cooked muscle tissue moisture, peptides/amino acids or PI (P> 0.45, ANOVA.). Sample size was considered too low to be of significance but four out of the six mushy crabs found during these experiments were from strings of pots pulled early in the day.

5.3.3 Transport, cooking, and chilling

There was considerable change in tissue firmness (TF) during the postharvest chain for both males and females (Fig 5.12a and b) with all crabs increasing in TF with cooking. Live males were firmer than live females upon arrival at the factory (P<0.001) but this difference disappeared upon cooking. Indeed, transport method (i.e. live or iced) and post-cook cooling method (slurry or chiller) had no significant impact on final cooked TF (Nested ANOVA, P>0.05).

Weight loss in whole crabs was also highly variable (Mean 17.63 + 0.76, Range: -4% to 50%, ) and changed significantly with transport and post-cooking cooling methods. Crabs that were slurried after cooking lost significantly less weight than those that were chilled (17.06 + 0.67 cf 11.27 + 1.38, P= 0.001, Fig 5.13) for both transport methods on most days. The % weight loss data from the 2nd, 4th and 6th of April were pooled as day/sex and transport method had no effect on weight loss (P= 0.832 and 0.718 respectively) for those days.

On the 8th of April iced females lost less weight than live crabs when chilled after cooking (P=0.001) (Fig 5.13b). Live females also lost significantly less weight if slurried rather than chilled after cooking (P=0.001); a similar reduction for iced crabs was not significant (P=0.162). Weight loss data from postmoult crabs were not included in analyses for treatment effects as this would have confounded the interpretation (ref Fig 5.14).

Fig 5.12 Tissue firmness (TF) in crabs at each stage of the cold chain from boat to chiller/freezer.

Males were put into a blast freezer then thawed for dissection the following day. Females were cooled in the chiller overnight after cooking/ice slurry. Significant differences between stages are indicated by dissimilar letters above columns relating to each date, black for 1/4/08, red for 4/4/08. Dates were analysed separately due to interaction.

6 a) Males 1/4/08 a 5 4/4/08 f ab ab f fg b efg 4

de TF 3 dg d c 2

c 1

0 On boatLive at Iced at Live after Live after Iced Iced factory factory cooker, cooker, after after chiller ice slurry cooker, cooker, chiller ice slurry All to blast freezer

6 b) Females 6/4/08 5 8/4/08 b b b b b b b 4 b

TF 3 a a a a 2 a

0 On boatLive at Iced at Live after Live after Iced after Iced after factory factory cooking cooking, cooking cooking, ice slurry ice slurry

All to chiller 39

Fig 5.13 % Loss in weight of whole crabs after cooking by date/sex for live vs. iced and chilled vs slurry treatments

Significant differences are indicated by dissimilar subscripts

Chilled Slurry

a) Males (2/4 and 4/4/08) and females (6/4/08) 20 a 16

% 12 b weight loss 8

b) Females (8/4/08) 20 a 16 % 12 weight loss 8 b 4 0 Live Iced

40 Two out of three postmoult crabs lost more weight than non-postmoult crabs from the same day/treatment (Fig 5.14). As indicated, one of these crabs was an extreme case (live females slurried, Fig 5.14), the other an outlier (Cooking trial) compared to other data for those experiments. Mushy crabs did not loose appreciably more weight than most of the other crabs from the same day/treatment. The same was true of 2 out of the 3 premoult crabs.

Biochemical parameters also changed with cooking but not with treatment (i.e. live/iced, chilled/ice slurry) (Figs 5.15 to 5.18). Females showed the most significant biochemical changes, presumably because they were placed in a chiller overnight, not blast frozen. Change in TF was not significantly linked (P>0.05) to polypeptide/amino acid concentration or PI within the range of concentrations recorded for these parameters in this study.

Fig 5.14 % Weight loss on cooking for premoult, postmoult and mushy crabs in comparison to % weight loss data for that period (box plots).

Pre and postmoult and mushy crab data are excluded from the boxplots. Refer Appendix 3 for explanation of box plots.

30 % Weight Loss 20

-10 November April Cooking trial 2007 2008 8/04/08

All data for that period

Live females: slurried

Iced males: chilled

Iced females: chilled

Mushy

Premoult

Postmoult

Postmoult and mushy

42 Fig 5.15 Muscle polypeptides/ amino acids (μg/g wet weight) of crabs at each stage of the cold chain from boat to chiller/freezer.

2000 a) Males 1/4/08 1600 4/4/08

1200 c μg/g c c 800 bc c

a ab 400

0 On boatLive at Iced at Live after Live after Iced Iced factory factory cooker, cooker, after after chiller ice slurry cooker, cooker, chiller ice slurry All to blast freezer

2000 b) Females 6/4/08 1600 8/4/08 c

1200

μg/g bc bc b b 800 b ab ab ab a a a 400 a

0 On On Live at Iced at Live after Live Iced Iced boat boat after factory factory cooking after after 4/4 6/4 cooking, cooking, cooking ice ice slurry slurry All to chiller

Fig 5.16 Muscle PI crabs at each stage of the cold chain from boat to chiller/freezer. Females were cooled in the chiller after cooking/ice slurry; males put into a blast freezer then thawed the following day. Significant differences are indicated by dissimilar subscripts, black for the first date of each pair, red for the second.

a) Males 1/4/08 100 4/4/08

PI 60 c c c bc c 40 aab 20

0 On boatLive at Iced at Live after Live after Iced Iced factory factory cooker, cooker, after after chiller ice slurry cooker, cooker, chiller ice slurry All to blast freezer

b) Females 6/4/08 100 8/4/08 c 80

PI 60 bc bc b b b 40 ab ab ab ab a a 20 a

0 On On Live at Iced at Live after Live Iced Iced boat boat factory factory cooking after after after 4/4 6/4 cooking, cooking cooking, ice ice slurry slurry

All to chiller 44

Fig 5.17 Total muscle protein (% wet weight) in crabs at each stage of the cold chain from boat to chiller/freezer.

25 a) Males 24 1/4/08 23 4/4/08 22 21 % 20 19 18 17 16 15 On boatLive at Iced at Live after Live after Iced Iced factory factory cooker, cooker, after after chiller ice slurry cooker, cooker, chiller ice slurry All to blast freezer

25 b) Females 24 6/4/08 23 8/4/08 22 21 a a ab % 20 ab ab ab 19 ab ab b b b b 18 b 17 16 15 On On Live at Iced at Live after Live Iced Iced boat boat factory factory cooking after after after 4/4 6/4 cooking, cooking cooking, ice ice slurry slurry All to chiller

Fig 5.18 Total muscle moisture (%) in crabs at each stage of the cold chain from boat to chiller/freezer.

Females were cooled in the chiller after cooking/ice slurry; males put into a blast freezer then thawed the following day. Significant differences are indicated by dissimilar subscripts, black for the first date of each pair, red for the second. a) Males 80 1/4/08 79 4/4/08 78 77 76 % 75 74 73 72 71 70 On boatLive at Iced at Live after Live after Iced Iced factory factory cooker, cooker, after after chiller ice slurry cooker, cooker, chiller ice slurry b) Females All to blast freezer 80 6/4/08 b 79 8/4/08 b ab b b b 78 ab ab ab 77 ab ab a ab 76 a % 75 74 73 72 71 70 On On Live at Iced at Live after Live Iced Iced boat boat factory factory cooking after after after 4/4 6/4 cooking, cooking cooking, ice ice slurry slurry All to chiller 46

5.3.4 Cooking trials

Whole crab weight loss was extremely variable during the cooking trials and highest at 100oC (P<0.001, Fig 5.19). Values were generally lower than those recorded from the commercial cooker. Tissue firmness and % moisture increased with temperature in both trials (P<0.001). Tissue PI and PPAA peaked at 80oC and declined slightly as temperature increased (Fig 5.20).

Fig 5.19 Percentage weight loss (+SE) in whole crabs during cooking trials on the 2-3/04/08 and the 6-7/04/08. Significant differences indicated by different coloured horizontal lines

16 2-3/04/08 14 12 6-7/04/08

10 % weight 8 loss 6

2 0

-2

-4 80 90 100

o Temperature ( C)

47 Fig 5.20 Cooking experiments: TF, % moisture, PI and PP_AA (+SE) for male crabs cooked at up to three temperatures (80, 90 and 100oC). Values from fresh uncooked crabs included (baseline). Significant differences indicated by different coloured horizontal lines or dissimilar superscripts. Data were pooled between dates for the analysis where there were no significant differences (P>0.05)

a) Tissue firmness (TF) 6 2/04/08

5 7/04/08

TF 3

0 Raw 80 90 100 Temperature (oC)

b) % Tissue Moisture 80 2/04/08

78 7/04/08

76 % 74

70 Raw 80 90 100 Temperature (oC)

c) Tissue PI 250 2/04/08

7/04/08 200 b

150 ab PI b ab 100 a c 50 a

0 Raw 80 90 100 Temperature (oC)

d) Tissue PP_AA 4000 2/04/08 3500 b 7/04/08 3000

2500 b μg/g 2000 b ab 1500 a b 1000

500 a

0 Raw 80 90 100 Temperature (oC)

49 Crab core temperatures did not reach the set water temperatures (Fig 5.21), as was also the case in commercial cooker trials (Fig 5.5)

Fig 5.21 Crab core temperatures during cooking at 80, 90 and 100oC

Cooking trial 1, 2/04/08.

100

80 Cooker temperatures (oC) 70 80

60 90 100 ToC 50 100 40

0 15:36 16:48 18:00 19:12 20:24 21:36 22:48 0:00

Time of day (24hr clock)

5.3.5 Hepatopancreatic protease

Hepatopancreatic protease activity was high and variable in crabs dissected on the boat (Fig 5.22). Cooking trials showed an almost complete deactivation after 13 mins at 90oC and no activity at 100oC. A regression (Fig 5.22b) based on the data shown in Fig 5.22 suggests remaining activity, after 13 minutes cooking, of approximately 18% at 80oC, 8% at 90oC and 0 at 100oC. Usual cooking temperatures are in the range 95-97oC, suggesting that the commercial cooking method is effective in reducing endogenous hepatopancreatic protease activity.

There were no significant differences in sex or day for any of these parameters (ANOVA, P>0.05).

51 Fig 5.22 Decline in hepatopancreatic protease activity (μg/g) with temperature

a) Representative data and regression line ( ). Protease activity = 354.027 – (4.880 x Temperature) + (0.013 x Temperature2), r2 = 0.855, P<0.001

360

320

280

240

200

Protease 160 Activity (μg/g) 120

0 0 102030405060708090100 Temperature (oC)

Baseline (fresh on boat) After cooking (1/4/08) males 1/4/08 Iced males cooked then chiller females 4/4/08 live males cooked then chiller males 4/4/08 live males cooked, slurry then chiller At factory Iced males cooked, slurry then chiller live males 1/4/08 Cooking Trials iced males 1/4/08 3/04/08 live males 4/4/08 7/04/08 Iced males 4/4/08

b) Decline in percentage protease activity with temperature. Based on regression derived from data shown in Fig 5.22a

100

60 % 50 Activity 40

0 20 30 40 50 60 70 80 90 100 Temperature (0C)

5.3.6 Mushiness

Eight crabs with poor flesh quality (i.e. mushy flesh) were detected during the project; i.e. 2.7% of the crabs cooked. An additional five crabs with mushy flesh were detected during the cooking trials. As these were all from the low temperature treatment (80oC), it is assumed that this was a treatment effect. TF readings from cooked mushy crabs were less than 3, below the range of values found in firm- fleshed crabs (3 to 5) (Fig 5.23).

Fig 5.23 shows the biochemical and physical data from these mushy crabs compared to that of non-mushy crabs sampled from the same day. The first 3 data sets (Fig 5.23 A to C) indicate a tendency for high poly-peptides/amino acids, and thus high PI, in mushy crabs compared to the distribution found in non-mushy crabs. Hepatopancreas protease levels for these crabs were similar to those found in firm crabs, suggesting that hepatopancreas protease is not responsible for the observed mushiness. During the April trip, hepatopancreas protease levels covered a broader range than those mentioned above but levels in the mushy crabs were also within the range of enzyme activity recorded from non-mushy crabs

Moisture levels were similar to those found in firm crabs unless the crabs were moulting in which case levels were high, up to and beyond the extremes found in the non-mushy crabs.

54 Fig 5.23 Biochemical profiles of crabs with mushy flesh (coloured symbols) compared with non-mushy crabs from the same day (box plots).

Individual crabs on a given day are indicated by same colour across parameters displayed. Red symbols (Figs C and E) indicate mushy postmoult crabs and for comparison a non-mushy postmoult crab (gold symbol) is included in Fig E. Figs D and E are crabs from 80oC treatment of cooking trials on that day. PP_AA = Polypeptides_ Amino Acids; PI = Proteolysis Index, TF = Tissue firmness. Refer Appendix 3 for explanation of box plots.

A) 14 11 07 (male)

84 7000 500 150

82 6000 400

80 5000 100 300 78 4000

76 3000 200 50 74 2000 100 72 1000

70 0 0 Moisture PP_AA PI Protease (%) (μg/g) (μg/g)

B) 19 11 07 (male) 84 7000 500 150

82 6000 400 80 5000 100 78 4000 300

76 3000 200 50 74 2000 100 72 1000

70 0 0 0 Moisture PP_AA PI Protease (%) (μg/g) (μg/g)

C) 21 11 07 (female)

84.0 7000 500 150

82.0 6000 400 80.0 5000 100 300 78.0 4000

76.0 3000 200 50 74.0 2000 100 72.0 1000

70.0 0 0 0 Moisture PP_AA PI Protease (%) (μg/g) (μg/g) D) 3 04 08 (male) Cooking trial (80oC treatment only)

84 7000 500 150 6.0

82 6000 5.0 400 80 5000 100 4.0 78 4000 300 3.0 76 3000 200 50 2.0 74 2000

100 1.0 72 1000

70 0 0 0 0.0 Moisture PP_AA PI Protease TF (%) (μg/g) (μg/g)

56 E) 8 04 08 (males). Cooking trial (80oC treatment only)

84 7000 500 150 6

82 6000 5 400 80 5000 100 4 78 4000 300 3 76 3000 200 50 2 74 2000 100 1 72 1000

70 0 0 0 0 Moisture PP_AA PI Protease TF (%) (μg/g) (μg/g)

5.3.7 Examination of the effect of snap freezing and liquid nitrogen storage on tissue levels of protease and peptide/free amino acids.

Paired T–Tests showed significant differences between fresh and frozen tissue for protease for 5/02/08 (Day 0) (P<0.013) (Fig 5.24) but not for peptides/free amino acids (P>0.05). Storage of dissected crabs on ice for 3 days significantly reduced protease (P<0.001) and increased peptides/free amino acids (p<0.001) relative to Day 0 levels. Stored crabs also showed substantial reductions in tissue integrity – raw muscle tissue was more friable and hepatopancreases had autolysed.

Fig 5.24 Effect of snap freezing and ice storage on hepatopancreas protease (μg/g) and muscle peptides/free amino acids (μg/g).

Dissimilar superscripts indicate significant differences (Protease - black letters, Peptides/AA – red letters)

180 1600 160 a b 1400 e 140 c c e 1200 120 1000 Protease 100 Peptides/AA (μg/g) 800 (μg/g) 80 600 60 d 40 d 400

20 200

0 0 fresh snap on on frozen ice ice/ snap

frozen 5/2/08 8/2/08 Date

5.3.8 Moult Stage/Shell Hardness

Eight premoult (all Stage D1) and 6 postmoult crabs (Stages A – C1) were found during the trials. Of these, 3 premoult and 3 postmoult crabs were cooked and the remainder dissected raw (i.e. baseline) during the experiments described previously. The remaining crabs were intermoult.

Of the parameters measured, TF showed the only obvious trends (Fig 5.25, rest of data (PI etc) not shown). The two cooked postmoult crabs had TFs near to or below the lower extreme of the cooked TF range for intermoult crabs. The lowest cooked postmoult TF corresponds to one of the mushy crabs mentioned earlier.

Fig 5.25 Relationship between TF in pre or postmoult (symbols) and intermoult (boxplots) crabs.

Refer Appendix 3 for explanation of box plots. 6

Texture 3

0 Raw Cooked

postmoult raw premoult cooked postmoult cooked

59 5.3.9 Histology

Two parasites have been provisionally identified from muscle tissue and haemolymph samples by histological methods. These are thought to be the dinoflagelate Hematodinium spp. (Fig 5.26a) and an as yet unidentified acanthocephalan (Fig 5.26b). The microsporidian Ameson spp (mentioned in the project proposal) was not found. Parasite identity is to be confirmed via PCR later in 2008.

Fig 5.26 shows apparently "necrotic" multilobate nuclei (red arrows) within haemocytes which are actually "normal" Hematodinium sp. The crab immune response is indicated by pink granulocytes (black arrows) and the extensive damage to the muscle fibres (green arrows) is evident (B. Jones, pers comm.)

Fig 5.26 Parasites found in blue swimmer crab muscle tissue. a) Hematodinium sp. The scale bar is 50 μm.

b) Acanthocephalan (as yet unidentified; green arrow). These were associated with nerve tissue. The scale bar is 50 μm.

During the first trip (November, 2007) 73% of samples (total n=71) were found to contain Hematodinium, with a further 14% for which identification was uncertain (Fig 5.27). Of those samples containing Hematodinium 10% were heavily infected, 27% showed evidence of muscle repair (muscle nuclei in tracks) and 21% also contained acanthocephalans. Overall, acanthocephalans were found in 21% of samples surveyed although occurrence is probably underestimated as this parasite was associated with nerve tissue, and not all samples would have contained this material.

Fig 5.27 Parasite load and associated pathology: November 2007

a) Percentage of samples containing Hematodinium sp. 80 70

60 50 % No Hematodinium sp. Infection (n=9), 40 Crabs Hematodinium sp. identification uncertain (n=10) 30 Hematodinium sp. confirmed (n=52) 20 10 0 Infection category

b) Percentage of Acanthocephalans, and percentage of samples showing some form of pathology, within each of the above categories 80 70 60 50 % Crabs 40 30 20 10 0 no change acanthocephalans Muscle nuclei in in nuclei Muscle tracks Infection/muscle necrosis Infection/muscle Heavy infection necrosis infection Heavy /muscle

62 Ninety-eight samples were taken on the second trip (April 2008) (Fig 5.28), 21% of these contained Hematodinium with a further 4% for which identification was uncertain. Of those with Hematodinium, 60% showed muscle necrosis, with or without acanthocephlans. Acanthocephlans were found in 25% of samples surveyed, again probably an underestimate.

Fig 5.28 Parasite load and associated pathology: April 2008

a) Percentage of samples containing Hematodinium sp.

80 70 60

50 No Hematodinium sp. Infection (n=72), % Crabs 40 Hematodinium sp. identification uncertain (n=4) 30 Hematodinium sp. confirmed (n=20) 20

10 0 Infection category

b) Percentage of Acanthocephalans, and percentage of samples showing some form of pathology, within each of the above categories 80 70 60

50 % 40 Crabs 30

10 0

no change Muscle necrosisMuscle acanthocephalans

Acanthocephalans necrosis and muscle 63 5.3.10 Parasite load and Biochemistry

Infection category did not significantly affect PI (P > 0.05, Fig 5.29a and b) or other biochemical parameters measured on either trip. The increase in variability in those samples containing acanthocephalans in Trip 1 is probably due to the low n (3) for that category.

Fig 5.29 Proteolysis index and infection category

A+H = Acanthocephalans plus Hematodinium sp a) Trip 1 (November 2007)

35 30

25 PI 20

15 10 5 0

A+H

No infection No Heavy infection Heavy nuclei in tracks) Hem (plus muscle (plus Hem acanthocephalans

b) Trip 2 (April 2008) Muscle nuclei in tracks

30 PI 20

10 0

A+H

sp and or and sp No infection

muscle necrosis muscle Acanthocephalans Hematodinium 64 There was a significant increase in muscle protease with presence of parasites in general (Fig 5.30, ANOVA, P=0.035). There were no differences between the protease levels recorded for the two trips (P.0.05) so the presence-absence data were pooled for the above analysis.

Fig 5.30 Parasite presence /absence and muscle protease activity (μg/g wet weight)

November 2007 (n=5,7) April 2008 (n=8,7)

20 Protease activity (ug/g wet 15 weight) 10

0 No infection Infection

65 6. DISCUSSION

The objectives for the project were:

1 Determine the principal sources of variation in flesh quality in Blue Swimmer Crabs in Shark Bay, Western Australia.

2 Develop and apply a postharvest and/or processing strategy designed to reduce variation in flesh quality

There are many potential causes of variation in blue swimmer crab flesh quality. These include crab moult stage, sex, size, the presence and species of parasites, and post harvest handling on board and cooking and chilling in the factory. Of the 450 crabs sampled during this project only 2.7% were mushy. This was considerably lower than the level expected (10-20%), it is however possible that two field-trips missed periods where levels of flesh quality deteriorated.

6.1 Crab-related issues

6.1.1 Moulting

Just prior to the actual moult a crustacean will begin to take up water, diluting the haemolymph, and increasing tissue moisture levels (Stewart and Li, 1969, Depledge and Bjerregaard, 1989, Mykles and Skinner, 1990, Musgrove and Geddes, 1995). During the subsequent intermoult period the fluid is replaced by muscle tissue as the animal grows within its new shell, and the hepatopancreas enlarges as nutrients are stored in preparation for the next moult.

Postmoult blue swimmer crabs become mushy upon cooking and have a reduced shelf life (Slattery, et al., 1989). This was suggested in the present study where two out of the three cooked postmoult crabs (as determined from swimming paddle morphology) presented as mushy on dissection. Unfortunately, one of these two was part of the 80oC treatment in the second cooking trial, so the mushiness could have been caused by undercooking. Premoult crabs did not present the same issue, a result also reported by Slattery et al (1989). Similar moult cycle effects on cooked flesh quality have been reported for Malaysian freshwater prawns (Macrobrachium rosenbergii) (Angel, et al., 1986). They found that cooked pre and postmoult prawns had poorer flesh quality than intermoult animals.

In northern Shark Bay, blue swimmer crabs moult all year round, although there are peaks from January – March and in May/June and July/August (P Jecks pers comm).

66 There are, however, no data on the actual relationship between the moulting peaks and flesh quality at the factory. Crabs are graded onboard, with very soft crabs picked out. Given the speed with which grading takes place, it is likely that firmer- shelled, but still postmoult, crabs would make it through to the factory, and through the next grading (at the conveyer) to the cooker.

The above two grading points are obvious candidates for removal of postmoult crabs via a simple shell squeeze test. Grading out postmoult crabs would reduce the general incidence of postmoult-induced mushiness, particularly during peaks of moulting activity.

A more rigorous moult stage grading regime on the boat and the factory would reduce flesh quality issues attributable to postmoult crabs. If graded out on the boat as too soft, the crabs could be returned to the sea to harden or transferred into floating corfs for a few days, or placed in on-board flow-through live tanks for hardening at the factory. The latter would require installation of on-shore recirculation tanks and feeding of crabs.

Such tanks could also allow a soft-shell crab product to be explored. A rigorous boat-based selection identifying late premoult crabs (see below) would have to be applied to properly standardise a soft-shell product. This would be followed by monitoring of moulting activity within holding tanks and picking of just-moulted crabs. Once again crabs would have to be fed.

Grading for postmoult stage before crabs are individually placed on the cooking conveyer would also reduce the mushiness problem but at that point the crabs are generally dead so further hardening/filling of the shell is not possible. The latter notwithstanding, grading for moult stage should be carried out at both points in the handling chain.

A very useful reference to practical moult staging of portunid crabs is offered by a website on the blue crab (Callinectes sapidus) http://www.bluecrab.info/molting.html. Chesapeake Bay (USA) crab fishermen use 10 stages to categorise their blue crabs (Zinski, 2006) vis:

• Green crab - 14 to 50 days prior to moult, depending on crab size. Very hard shell.

• White sign peeler - Two weeks prior to moult.

67 • Pink sign peeler - One week prior to moult.

• Red sign peeler - Two days prior to moult.

• Rank peeler - Hours prior to moult.

• Buster - In the process of shedding its old shell.

• Soft shell - Immediately following moult.

• Paper shell - 12 hours after moult. Slightly stiff shell.

• Buckram - 24 hours after moult. Semi-stiff, crinkly-hard or leathery shell.

• Whitey - Four or more days after moult. The ventral surface of the shell is a lustrous white.

Fig 6.1 Mid moult cycle and peeler stages for the blue crab Callinectes sapidus. Sequence left to right: mid-cycle, white sign, pink sign, red sign. Photo: Alicia Young-Williams, Smithsonian Environmental Research Center

The white, red and pink sign mentioned above refers to the physical changes evident in the margins of the swimming paddles as the new shell develops under the old in readiness for a moult. It is reasonable to assume that similar signs would be visible during the premoult period in blue swimmer crabs – the premoult crabs found in the present study were all early “white sign peeler” stage – the old exoskeleton had just begun to separate and the new shell develop underneath. Further work is needed to refine these stages and their durations for the blue swimmer crab.

All crabs, including blue swimmers, cease feeding just prior to, during and immediately after moulting (Kangas, 2000, Stehlik, et al., 2004). As the crab’s shell

68 hardens feeding begins again, with peaks during early intermoult and late premoult. The length of the non-feeding period will vary with species, size and temperature and will affect catchability. Using the above terminology this means that it would be rare to find crabs from rank peeler through to softshell (only one, a softshell, was found by the authors during the project) but harder-shelled crabs would come into pots. Specific differences notwithstanding, shell hardening after the moult may be faster than indicated in Chesapeake Bay (36o55’N) as Shark Bay (24o30’S) is closer to the tropics (23o26’S to 23o26’N) but at peak moulting times enough postmoult crabs may come into the pots to make mushy flesh a significant issue.

It is acknowledged that extra grading may impact on processing chain efficiency. However, with practice the impact would be minimized, with any negative impact balanced by removal of potentially mushy crabs from the product line. The durometer used in this study could be utilised to standardise squeeze-test measurements.

6.1.2 Parasites

Histological analysis suggested the presence of two parasites, acanthocephalans (thorny-headed worms), genus unknown and Hematodinium sp, a dinoflagellate blood parasite.

Acanthocephalans live in crustaceans and insects as juveniles and vertebrates as adults; the primary host is therefore the vertebrate (Schmidt and Roberts, 1981). A primary host is one in which the parasite reaches maturity and, if applicable, reproduces sexually. A secondary host harbors the one or more stages of the immature parasite until eaten by the primary host (Schmidt and Roberts, 1981). Species of acanthocephalan have caused mass mortalities in primary-host populations (i.e. common eider duck, Somatera mollissima, Camphuysen, et al., 2002) but the main effect on the secondary host appears to be that of behavioural change, making them more susceptible to the primary host/predator. This strategy has been suggested for three species of New Zealand shore crabs (secondary hosts for two acanthocephalan spp) (Latham and Poulin, 2002). A similar phenomenon has been reported for other acanthocephalan-secondary host relationships (e.g. the shore crab Hemigrapsus crenulatus with the acanthocephalan Profilicoulis antarcticus, Haye and Ojeda, 1998). In the present study, the parasite was always found associated with nerve tissue in the muscle of the swimming paddle muscle

69 mass. Although not tested, it is suggested that, at high infestation levels the function of the paddle could be impaired, making the crab more susceptible to predation by the primary host, possibly a shark or large fish. The main effect may thus be an increase in blue swimmer crab mortality with rising infestation levels. It is also possible that the integrity of the muscle may become increasingly compromised with infestation level, affecting flesh quality. It is worthy of note that the sampling technique used in this study probably under-estimated acanthocephlan incidence as not all muscle tissue samples would have necessarily contained nerve tissue.

Hematodinium spp primary hosts are marine decapod crustaceans. These include commercially important species such as Norway lobster (Nephrops norvegicus) off the coast of Scotland (Stentiford et al 2000) snow and tanner crabs (Chionectes opilio and C. bairdi) off Alaska and western Canada and blue crabs (Callinectes sapidus) from Chesapeake Bay (eastern USA) (Stentiford and Shields, 2005). A Hematodinium-like dinoflagellate impacts on the edible crab (Cancer pagurus) in UK waters (Stentiford et al 2002) and H. australis has been reported from the mud crab (Scylla serrata) (Hudson and Lester, 1994) and the blue swimmer crab (Portunis pelagicus) (Shields, 1992, Hudson and Lester, 1994) off the east coast of Australia (Moreton Bay). Hematodinium spp invade and spread throughout the haemolymph, musculature, hepatopancreas and other organs and frequently cause host mortality (Shields, 1992, Hudson and Shields, 1994, Stentiford and Shields, 2005). Seasonal outbreaks (called epizootics) of these parasites have significantly damaged commercial stocks of all the abovementioned northern hemisphere species (Stentiford and Shields, 2005) with mortalities mainly occurring in the unfished juveniles and females (Stentiford and Shields, 2005).

In the present study, little can be said with regard to seasonality because the November sample was taken on western side of Shark Bay, and the April sample taken on the eastern side, 30km south of Carnarvon (Fig 4.1). That stated, 73% (n=71) of crabs (males and females equally) were found to be infected with Hematodinium in November 2007 and 21% (n=96) in April 2008, these numbers are considerably higher than those reported for Hematodinium in Moreton Bay (Queensland) mud crabs (1.5%, n=130, Hudson and Lester, 1994) and blue swimmer crabs (0.9, (n=205) to 4% (n unknown), Hudson and Shields, 1994). The apparently high sublethal incidence of the parasite on both sides of Shark Bay may indicate a

70 large pool of the parasite in the bay, and therefore the potential for epizootic events, but this awaits further study.

The effect of Hematodinium spp. on the flesh varies with species of crabs and of parasite, although a common feature of crabs in advanced stages of Hematodinium infection is hyper-pigmentation of the crab carapace, often accompanied by a chalky or cooked appearance and discolouration of membranes at the leg joints (Stentiford and Shields, 2005). The haemolymph at this stage is opaque to creamy.

There are several variants. For example, the Alaskan tanner crab (C. bairdi) and the snow crab (C. opilio) are prone to infection by a form of Hematodinium (as yet undescribed to species level) which causes Bitter Crab Disease (BCD) (Meyers, et al., 1987, 1990, Pestal, et al., 2003, Shields, et al., 2005). As the name suggests, infected crabs often have bitter meat, which is also of irregular texture (Shields et al. 2005). BCD is therefore responsible for significant economic losses in the above crab fisheries. A slightly different pathology is displayed by another Hematodinium sp which, in this case, causes Pink Crab Disease (PCD) in both the tanner crab (Stentiford, et al., 2002) and the Norway lobster (Stentiford, et al., 2000). The carapace and appendages of infected crabs become hyper-pigmented. Harvested crabs with the disease show reduced survival during holding and transport, and the yield, texture and appearance of the meat are negatively impacted (Stentiford, et al., 2002). The blue crab (C. sapidus) is prone to infection by Hematodinium perezi (Stentiford and Shields, 2005); the heavily infected crabs are sluggish and opaque muscles can be seen through the ventral carapace, or the carapace is pink (Messick, 1994)

The blue swimmer crabs in the present study showed none of the gross signs of infection (shell or tissue) described above. In one case the haemolymph of a crab was milky but histology suggested that this was a bacterial infection not a dinoflagellate and there was no evidence of Hematodinium found in the corresponding tissue sample.

The Hematodinium diagnosis is supported by the histological studies although the authors await confirmation by PCR analysis, currently underway. Crabs in the advanced stages of the disease may not have survived to be caught, either as a result of predation or disease-induced mortality; although one might expect at least some external signs suggesting degrees of infection in live crabs. There was,

71 however, clear tissue pathology (focal infections, necrosis, signs of tissue repair) associated with the presence of the parasite. There was also the significant increase in muscle protease activity in infected tissue. The protease responsible was probably of crab, not parasite, origin. Little work has been done on the enzymes of Hematodinium spp however the only enzyme isolated from H. perezi infecting C sapidus has been leucine arylamidase (Small, et al., 2007). This enzyme breaks down the amino acid leucine into acetyl acetate and acetyl Co-A and its activity would not be picked up by the protease kit used in the present study, which utilised fluorescene-tagged casein as a substrate (P Babidge, pers. com.). However the elevated protease level in the infected tissue may still indicate parasite activity. Stentiford et al (2002) suggested that severe disruption of the muscle during PCD in C. pagurus could assist in activation of proteases responsible for normal premoult muscle atrophy in crustaceans, as described by Mykles and Skinner (1990). This would benefit the parasite by provision of a ready source of nutrients.

It is also possible that crabs were sampled at low points in the parasite’s activity cycle; as other Hematodinium spp have been described as highly seasonal with “lows” showing almost no activity (Stentiford and Shields, 2005). It is possible that what was found in this study was actually a low, in terms of virulence. If this was the case, a much higher level of the parasite (i.e. an epizootic) might be expected to occur occasionally, with concomitant effects on mortality and on flesh quality of cooked crabs. There would be little that could be done to prevent such an event.

6.1.3 Mitigation

A flesh quality monitoring program may quantify changes in flesh quality, and would be equally useful in identifying such a problem originating from postmoult crabs. This would allow a measure of damage control by reducing contamination of good product. The monitoring program could take the following form:

1. Train the crab meat pickers as to what is acceptable and what is not with regard to flesh quality. This would be a simple texture test done by hand/eye on each crab as it is picked up for flesh removal.

2. Crabs which are unacceptable are put out to one side and rechecked (by another picker) at the end of the picking session. They are either included in the general picked meat or discarded. The penetrometer used in this study could be

72 utilised to standardise flesh quality between pickers, with a reading of less than 3 corresponding to “mushy”.

3. A daily tally is kept of crabs with poor flesh quality. This program would pick up seasonality, moult periodicity, gender and size effects and would improve control over flesh quality. It would also provide a better economic impact estimate.

This method is obviously not available for those crabs (A-grade males and females) which are not picked but cooked, frozen whole and sent to market. Further parasitological studies are needed, with information required on including variation in prevalence by area and season, and external signs which may be useful in grading before cooking. In this regard, some of the signs discussed above for other species may be useful. In the interim, the presence of mushy flesh in picked crabs should indicate the problem in the incoming crabs in general.

6.2 Post harvest handling

There were no differences in tissue texture (or biochemistry) with onboard postharvest treatment (i.e. live vs iced) or with post-cooking cooling method (ice slurry vs chiller). There were also too few mushy crabs found to examine the frequency of this condition under the above treatments. The paucity of mushy crabs suggests that present postharvest handling methods were adequate under the conditions found during the two field trips (if 2.7% is an acceptable mushiness level) but that during periods when tissue quality is of the order suggested by industry (10- 20%), extra measures may be necessary:

6.2.1 On-board handling

Crabs die quickly when put on ice; ice cover must be maintained over the whole catch (including the top crabs in a crate) to maintain freshness. Other useful measures could include reflective insulated covers or tarpaulins for covering crates stored on-deck or on the cabin roof. Extra insulated bins/chillers would be the best solution to the above, however deck space is limited.

6.2.2 Green crab chiller

The green crab chiller’s (GCC) function is compromised by continual opening and closing of the outer door when the crabs arrive off the boat, then the inner door (Fig 6.1) as they are loaded onto the conveyer for cooking. While the placement of an

73 anteroom (Fig 6.2) to insulate the chiller from the factory is not spatially or logistically practical, such a room, if placed to insulate the chiller from the outside, would maintain a lower and more stable temperature, reducing the load on the chiller and therefore running costs. Such a reduction would be partly due to a lower input of humid air from the outside, limiting frosting on the chiller room evaporator coils. This would reduce both the time the refrigeration plant has to run to maintain the temperature and the number of defrosting cycles needed per day.

74 The anteroom would be relatively small and therefore could be cooled with a small chiller unit. Crabs could be loaded into the anteroom, the door closed, and when the temperature is at or near that of the GCC, moved into that room. Note that the outside door of the anteroom is placed perpendicular to that of the GCC to limit the loss of cold air from the GCC when the rooms are in use.

Fig 6.1 Existing Abacus Crab Factory (not to scale)

FreezersStore

Green Crab -28C -22C Chiller

A Cooked Crab Chiller Packer

Reject Packer Tearoom bins

Wash Weighing station Weighing room / WC Office

Store

B Cooker C Shrink wrapper

A = Green crabs loaded B = Crabs transferred into cooker from conveyer via tipping bucket C = Cooked crabs into hopper then down Picking Store Store Room to manual sorting station Conveyor

Green crabs

Cooked crabs Sliding doors Swinging doors

75 Fig 6.2 Suggested modifications to the green crab chiller

Outside Outside

Anteroom 21

Green Crab

Existing Green Crab wall Chiller Chiller

Chiller

Factory

Addition

Existing chiller unit

Additional chiller unit

Existing sliding door Additional sliding door

Plastic strip curtain

76 6.2.3 Cooking

Muscle tissue firmness and tissue breakdown products (i.e. polypeptides/amino acids) increase; proteases are largely inactivated and crab weight is reduced during cooking.

Crabs were only kept overnight before sampling. It is possible that even the residual enzyme activity measured in this study could affect tissue quality if crabs were thawed by customers and kept chilled for any length of time. The impact of this may have been reduced during the first fieldtrip when the cooker temperature was increased from 95oC to 97oC, on the author’s suggestion.

It is assumed that weight reduction is due to loss of body fluids. Mean weight loss on cooking (17.6%) was about the same as that reported for cooked spiny lobsters (15%, Panulirus argus and P laevicauda, OgawaI, et al., 2007) and slightly higher than found in dungeness crabs (12.5%, Cancer magister, Hackett, et al., 2003). Undetected damage during grading or ruptures in joints during cooking may account for some of the higher values (i.e. 50%). Postmoult crabs generally appeared to lose proportionally more weight than intermoult animals, presumably because their tissue contains more fluid, as discussed earlier. The hepatopancreas disintegrates during cooking but the remains may not necessarily be lost from the crab body, unless the crab is damaged as suggested above.

Slattery et al (1989) reported an increase in shear force (similar to tissue firmness in the present study) and texture grade and a decrease in PI with cooking time. Similar trends were observed with increasing temperature in the present study. Lower temperatures increased PI but at the same time the highest temperature (100oC) caused the greatest weight loss. During cooking trials all crabs were chilled immediately in ice slurry which also reduced weight loss in trials using the commercial cooker.

The major issues may be the length of time crabs spend in hot water under 80oC and the temperature differential between crabs going into the cooker and the set cooker temperature. This is especially evident for the highest temperatures.

As the hepatopancreas disintegrates enzymes would spread throughout the flesh, with concomitant proteolytic activity causing tissue breakdown, the rate increasing with temperature up to 80oC. This is particularly evident in the 80oC treatment where

77 the crab core (Fig 5.22) did not reach the proteolytic enzyme deactivation temperature reported by Slattery et al (1989). The same may apply to enzymes of parasitic origin; Hematodinium or acanthocephalan enzymes already in the flesh may increase in activity with temperature, contributing to flesh breakdown. As discussed earlier, the enzyme leucine arylamidase has been isolated from Hematodinium perezi (infecting the blue crab C sapidus, Small, et al., 2007). It is unknown whether the Hematodinium identified in this study carries that enzyme, however a cactus (Opuntia ficus-inidica) extract containing leucine arylamidase showed maximum activity at 55oC with a minima at 0 and 70oC (at pH 5.2, Teixeira, et al., 2000).

At present, crabs are going from the green crab chiller at 2-4oC and entering 97oC water. This large thermal shock for the crabs appears to influence the level of weight loss. The steam injection system used by Abacus Fisheries is efficient; however, the authors would suggest trialling a pre-warming step in the process to alleviate thermal shock effects on the tissue and to reduce cooking time. Iced (and usually dead) crabs coming from the green crab chiller could be placed in room temperature seawater for long enough to increase the crab core temperatures to ambient (time will depend on load) before placing in the cooker. This would reduce the thermal shock and by decreasing the required cooking time/time to 80oC possibly reduce tissue breakdown. This same idea is used with success in shrimp-cooking factories in India (M Raj, pers comm). Shrimp are sent through a steam injection cooker (eg. http://www.dantech.com.sg/) with variable temperatures, increasing with distance into the cooker. Cooking time is 2 to 4 minutes, in a steam/water spray (no immersion).

This study showed that placing cooked crabs in ice slurry reduced weight loss. These crabs were drained overnight after slurrying, so captured water was not an issue. It is recommended that crabs be placed in ice slurry after cooking to remove heat as quickly as possible. They could then be placed in the postcook chiller for draining and to maintain cold core temperatures

6.2.4 Post Cook Chiller

Cooling times should be reduced as much as possible. At present, cooling to 0oC after cooking takes between 4.5 and 13.5 hours, if 0oC is reached at all.

The post-cook chiller has sliding doors which open directly to the factory and the outside (Fig 6.1). As a result there are wide fluctuations in chiller temperature (Fig

78 5.10) as these doors are periodically opened and closed (the chiller is often used as a thoroughfare), and the chiller is loaded with cooked crabs. This usage facilitates the entry of ambient, humid air (and potentially dust) into the processing area. In addition, the wide variation in storage temperature could damage the texture of product stored in the chiller.

The desired temperature (0-4oC) is maintained for a relatively short time, if at all, with staff placing hot crabs in the chiller in the afternoon and picked meat in there the following morning (Fig 5.10).

It is suggested that two anterooms are added to the post-cook chiller to maintain its temperature. This will remove the chilling, cooked product from the air space subject to fluctuation caused by opening the factory and outside doors (Fig 6.3). Smaller air spaces are also easier to chill and limit temperature fluctuations.

Crabs should be put into Anteroom 1 for draining and further chilling after cooking and initial cooling in ice slurry or other form of cooling bath. Once the anteroom reaches capacity, crabs could be moved into the chiller proper. This should be done with the factory-side door closed which should maintain the temperature in the chiller and reduce the load on the chiller unit. Air exchange between the anteroom and the chiller or between the factory and the anteroom could be limited further with a plastic strip curtain, as used on both chillers’ outside doors.

The same process could be carried out with Anteroom 2 (Fig 6.3): load with crabs, close the inner door then open the outer door to remove the product. This may work more efficiently than the present plastic strip curtain, the efficiency of which may be compromised by passage of staff from the outside. Anteroom 2 could also act as a secondary chiller when extra capacity is required (hence the suggested second chiller unit).

79 Fig 6.3 Suggested modifications to cooked crab chiller (not to scale) Outside

Anteroom 2

Existing wall Cooked Crab Chiller

Partition

Factory Anteroom 1

Office

Addition

Existing chiller unit

Additional chiller unit

Existing sliding door Additional sliding door Plastic strip curtain

7. BENEFITS AND ADOPTION

Adoption of the recommendations contained in this report will improve flesh quality and processing efficiency for the blue swimmer crab fishery based in Carnarvon, WA. This project has been carried out in close collaboration with Abacus Fisheries and it is anticipated that the recommendations herein will be adopted where possible. Discussions during the project have already resulted in changes to factory operations (eg. raising of cooker temperature)

The company has a national profile, having won a number of awards for its products. Such a profile will facilitate the dissemination of recommendations made within this report to other vertically integrated crustacean fishing companies in Australia.

8. FURTHER DEVELOPMENT

Further discussions will be held with Abacus Fisheries, at need, regarding the adoption of recommendations, particularly the monitoring program.

Further research will be carried out to develop a practical field test for the Hematodinium. Work has begun on an ARC-linkage grant with top-up funding to be sought from FRDC and other sources.

The penetrometer was very useful in the measurement of blue crab flesh quality and will be developed further for use on other species.

The durometer was useful for field measurement of shell hardness. Further work should be conducted on blue swimmer crabs to better understand moulting periodicity and moult-stage related effects on flesh quality. The durometer’s usefulness will also be evaluated for other species (eg southern rock lobster).

9. PLANNED OUTCOMES

The planned outcomes were:

1. An understanding of the sources of variation in flesh quality in blue swimmer crabs in Shark Bay Western Australia.

2. A harvest and processing strategy linked to an understanding of seasonal variation of flesh quality.

3. Uniform consistency in product quality aligned to the desired premium market positioning for blue swimmer crabs sold by Abacus Fisheries Pty Ltd and other

81 companies.

Several issues have become apparent with regard to sources of variation in flesh quality, particularly with regard to the presence of parasites, and their impact on flesh quality; the impact of the moult cycle stage on post-cooking flesh quality and the likely impact of cooking time, time to 80oC and post cook chilling on flesh quality

A monitoring program has been recommended to determine the seasonality of exogenous effects (i.e. moult stage, parasites) on flesh quality. This information will improve understanding of seasonal variation in flesh quality and thus facilitate appropriate modifications in harvest and processing strategies. An understanding of exogenous effects, and uptake of the other recommendations regarding crab processing will lead to a more uniform consistency in blue swimmer crab flesh quality.

10. CONCLUSION

The objectives of the project were:

1 Determine the principle sources of variation in flesh quality in Blue Swimmer Crabs in Shark Bay, Western Australia.

2 Develop and apply a postharvest and or processing strategy designed to reduce variation in flesh quality

Several issues have become apparent, particularly with regard to:

• The presence of parasites, and their impact on flesh quality. Two groups of parasites were provisionally identified using histological methods (Hematodinium spp and Acanthocephalans) with the former known to have significant impacts on flesh quality and mortality in other crab fisheries around the world.

• The impact of the moult cycle stage on post-cooking flesh quality. Postmoult crabs have reduced texture on cooking due to an increase in flesh moisture content.

• The likely impact of cooking time, time to 80oC and post cook chilling on flesh quality

The impact of parasites and postmoult crabs on post-cooking texture may also be monitored and its effect reduced by removal of mushy crabs. This is of particular importance as meat from one mushy crab would spread through the picked meat, having a broader impact than simple numbers might suggest. The penetrometer used in this study could be utilised to standardise flesh quality between pickers, with a reading of less than 3 corresponding to “mushy”. The method is obviously not available for those crabs (A-grade males and females) which are not picked but cooked, frozen whole and sent to market. Further parasitological studies are needed, with information required on including variation in prevalence by area and season,

83 and external signs which may be useful in grading before cooking. In this regard, some of the signs discussed above for other species may be useful. In the interim, the presence of mushy flesh in picked crabs should indicate the problem in the incoming crabs in general.

Suggestions are made for alterations to boat and factory infrastructure, particularly cooking and cooling processes. Onboard handling could be improved, particularly with regard to the present storage of crabs on deck once conventional storage bins are full. Reducing cooking time by pre-warming crabs and ice slurrying after cooking would improve cooking efficiency, reducing tissue breakdown and crab weight loss. In addition, limiting connectivity between the factory/outside and the chiller would reduce the chiller unit workload/running costs and maintain product quality.

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APPENDIX 1: INTELLECTUAL PROPERTY

There are no intellectual property issues arising from this research.

APPENDIX 2: STAFF

Dr Richard Musgrove

Mr Steve Slattery

Mr Peter Jecks

Professor Brian Jones

APPENDIX 3 BOX PLOTS

Box plots are used in Fig 5.14, 5.23 and 5.25. The size of the box indicates the spread of the central 50% of the data. The position of the median in relation to the centre of the box indicates how skewed (i.e. asymmetrical) the data are. In the example below, the data are skewed toward lower values.

Extremes: Values which are greater than 3 times the box height from the 3000 upper or lower quartile Outliers: Values which are 1.5 – 3 times the box height from the upper or lower quartile 2000 Whiskers: Greatest or smallest non- outlier value Upper quartile: 25% of the data are 1000 greater than this value

Median: the middle of the data set Lower quartile: 25% of the data are 0 less than this value

PP_AA

APPENDIX 4 CALIBRATION CURVE FOR PENETROMETER USED IN TISSUE QUALITY ANALYSIS. Load applied (g) vs Penetrometer reading (PR)

4000 Load (g) = 47.068 x PR + 400.83 3500 R2 = 0.9992 3000

2500 g 2000

1500

1000 500

020406080

Penetrometer reading

Raw data were divided by sample weight (g) and expressed as Tissue Firmness (TF)

90 Innovative Food & Plants

Empowering Industry R&D: Uniform flesh quality for premium market positioning of blue swimmer crabs

Project No. 2007/244

R. Musgrove and S. Slattery

January 2009

Government of South Australia PP_090027