Microbiology Handbook Fish and Seafood

The Microbiology Handbook- Fish and Seafood consists of the microbiology of seven different product categories: chilled and frozen raw fish, chilled andfrozen prepared fish, molluscan sh

Leatherhead Food International

This edition first published 2009 by

Leatherhead Publishing, a division of

Leatherhead Food International Ltd

Randalls Road, Leatherhead, Surrey KT22 7RY, UK

URL: http://www.leatherheadfood.com

and

Royal Society of Chemistry

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Cambridge, CB4 0WF, UK

URL: http://www.rsc.org

Regstered Charity No 207890

ISBN: 978-1-905224-76-0

A catalogue record of this book is available from the British Library

© 2009 Leatherhead Food International Ltd

The contents of this publication are copyright and reproduction in whole, or in part, is not permitted without the written consent of the Chief Executive of Leatherhead International Limited.

Leatherhead International Limited uses every possible care in compiling, preparing and issuing the information herein given but accepts no liability whatsoever in connection with it.

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as permitted under the terms of the UK Copyright, Designs and Patents Act, 1988, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of the Chief Executive of Leatherhead International Ltd, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licencing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK Enquiries concerning reproduction outside the terms stated here should be sent to Leatherhead International Ltd at the address printed on this page.

Printed and bound by Biddles Ltd., King’s Lynn

Associate Prof Covadonga Arias

The Grimsby Institute

Humber Seafood Institute

Pacific Regional Laboratory Northwest

U.S Food and Drug Administration

University of SurreyGuildford

Surrey GU2 7XHUnited Kingdom

Jeffrey L.C Wright C.M., Ph.D.,FCIC

Carl B Brown DistinguishedProfessor of Marine ScienceUNCW Center for Marine ScienceMarvin Moss Lane

Wilmington

NC 28409United States of America

Rhea Fernandes and Dr Peter Wareing Leatherhead Food InternationalRandalls Road

LeatherheadSurreyKT22 7RYUnited Kingdom

Eugenia Choi and Dr Jenny PflegerRegulatory Advisors

Leatherhead Food InternationalRandall Road

LeatherheadSurreyKT22 7RYUnited Kingdom

The Microbiology Handbook series includes Dairy Products, Fish and Seafood,and Meat Products, published by Leatherhead Food International and RSCPublishing The books in the series are designed as easy-to-use guides to themicroorganisms found in foods Each book provides a brief overview of theprocessing factors that determine the nature and extent of microbial growth andsurvival in the product, potential hazards associated with the consumption of arange of products, and growth characteristics for key pathogens associated withthe product All handbooks also contain a review of the related legislation inEurope and UK, guides to HACCP, and a detailed list of contacts for various foodauthorities The books are intended as a source of information for microbiologistsand food scientists working in the food industry and responsible for food safety,both in the UK and elsewhere

I dedicate this book to my husband - Goldwyn; thank you for all the support

Rhea Fernandes Leatherhead Food International

5 CURED, SMOKED AND DRIED FISH 93

5.3 Processing and its Effects on the Microflora 96

7.7 Neurotoxin Shellfish Poisoning (NSP) Toxins 153

8.4 Implementation and Review of the HACCP Plan 184

Fish and seafood are a main source of animal protein in the diet Because of theirhealth advantages over red meats, the consumption of fish and seafood hasincreased Catches can be gathered from seas, rivers and lakes whose water canrange from pristine to contaminated Often contamination is from human andanimal sources; thus, fish and seafood can be involved in the transmission ofpathogenic microorganisms and toxins Geographical region, season, and, for fish,whether they are pelagic (surface to mid-water) or demersal (bottom) feeders willinfluence the numbers and types of microorganisms present on freshly caughtseafood

The Microbiology Handbook- Fish and Seafood consists of the microbiology

of seven different product categories: chilled and frozen raw fish, chilled andfrozen prepared fish, molluscan shellfish, crustacean shellfish, cured, smoked anddried fish, fermented fish, and fish and shellfish toxins The second edition of thishandbook is a review of the entire book for currency of information Key changes

in this edition are the recent regulatory changes pertaining to food hygiene andmicrobiological criteria for foodstuffs

Further Reading

General Fish/Seafood Microbiology

Cross N Seafood processing: basic sanitation practices, in Handbook of Food Products Manufacturing: Health, Meat, Milk, Poultry, Seafood and Vegetables Eds Hui

Y.H., Chandan R., Clark S., Cross N.A., Nollet L.M Hoboken, Wiley 2007, 67.

947-Luten J.B., Jacobsen C., Bekaert K., Saebo A., Oehlenschlager J Seafood Research from Fish to Dish: Quality, Safety and Processing of Wild and Farmed Fish.

Wageningen, Wageningen Academic Publishers 2006.

International Commission on Microbiological Specifications for Foods Fish and fish

products (Microorganisms in fish products), in Microorganisms in Foods 6: Microbial Ecology of Food Commodities Ed International Commission on

Microbiological Specifications for Foods New York, Kluwer Academic / Plenum Publishers 2005, 174-249

Blech Z.Y The fish industry, in Kosher Food Production Ed Blech Z.Y Oxford,

Blackwell Publishing 2004, 167-77.

Stanfield P Seafood: processing, basic sanitation practices, in Food Processing:

Principles and Applications Eds Smith J.S., Hui Y.H Oxford, Blackwell

Publishing 2004, 459-72.

Stanfield P Seafood processing: basic sanitation practices, in Food Plant Sanitation Eds.

Hui Y.H., Bruinsma B.L., Gorham J.R., Nip W-K., Tong P.S., Ventresca P New York, Marcel Dekker 2003, 543-61.

Gudmundsson M., Hafsteinsson H Minimal processing in practice: seafood, in Minimal Processing Technologies in the Food Industry Eds Ohlsson T., Bengtsson N.

Cambridge, Woodhead Publishing Ltd 2002, 245-66

Bremner H.A Safety and Quality Issues in Fish Processing Cambridge, Woodhead

Publishing Ltd 2002.

Heinitz M.L., Johnson J.M The incidence of Listeria spp., Salmonella spp., and

Clostridium botulinum in smoked fish and shellfish Journal of Food Protection,

1998, 61 (3), 318-23.

Dalgaard P Predictive microbiological modelling and seafood quality, in Seafood from Producer to Consumer: Integrated Approach to Quality; Proceedings of the International Seafood Conference, Noordwijkerhout, November 1995 Ed Luten

J.B Amsterdam, Elsevier 1997, 431-43.

Huss H.H., Dalgaard P., Gram L Microbiology of fish and fish products, in Seafood from Producer to Consumer: Integrated Approach to Quality; Proceedings of the International Seafood Conference, Noordwijkerhout, November 1995 Ed Luten

Davies A.R Modified-atmosphere packaging of fish and fish products, in Fish

Processing Technology Ed Hall G.M London, Blackie 1997, 200-23.

Rawles D.D., Flick G.J., Martin R.E Biogenic amines in fish and shellfish, in Advances

in Food and Nutrition Research, Vo1ume 39 Ed Taylor S.L London, Academic

Press 1996, 329-65.

Gram L., Huss H.H Microbiological spoilage of fish and fish products International Journal of Food Microbiology, 1996, 33 (1), 121-37.

Ashie I.N.A., Smith J.P., Simpson B.K Spoilage and shelf-life extension of fresh fish and

shellfish CRC Critical Reviews in Food Science and Nutrition, 1996, 36 (1-2),

87-121.

International Commission on Microbiological Specifications for Foods Seafood toxins of

microbiological origin, in Microorganisms in Foods, Volume 5: Microbiological Specifications of Food Pathogens Ed International Commission on

Microbiological Specifications for Foods London, Blackie 1996, 265-79 Dalgaard P Qualitative and quantitative characterization of spoilage bacteria from packed

fish International Journal of Food Microbiology, 1995, 26 (3), 319-33.

Dalgaard P Modelling of microbial activity and prediction of shelf-life for packed fish.

International Journal of Food Microbiology, 1995, 26 (3), 305-17.

Sofos J.N Microbial growth and its control in meat, poultry and fish, in Quality

Attributes and Their Measurement in Meat, Poultry and Fish Products Eds.

Pearson A.M., Dutson T.R Glasgow, Blackie 1994, 359-403.

Ward D.R Microbiological quality of fishery products, in Fisheries Processing:

Biotechnological Applications Ed Martin A.M London, Chapman and Hall 1994,

1-17.

Gibson D.M Fish spoilage and safety PHLS Microbiology Digest, 1994, 11 (2), 118-20 Bonnell A.D Microbiological indicators and seafood processing, in Quality Assurance in Seafood Processing: A Practical Guide Ed Bonnell A.D London, Chapman &

Hall, 1994, 90-106.

Garrett E.S A new look at microbiological standards, guidelines, and specifications for

seafood products Activities Report of the R&D Associates, 1994, 46 (1), 54-61.

McMeekin T.A., Ross T., Olley J Application of predictive microbiology to assure the

quality and safety of fish and fish products, in Quality Assurance in the Fish Industry: Proceedings of an International Conference, Copenhagen, August 1991.

Eds Huss H.H., Jakobsen M., Liston J Amsterdam, Elsevier 1992, 459-78.

Gram L Evaluation of the bacteriological quality of seafood, in Quality Assurance in the Fish Industry: Proceedings of an International Conference, Copenhagen, August

1991 Eds Huss H.H., Jakobsen M., Liston J Amsterdam, Elsevier 1992, 269-82 Liston J Bacterial spoilage of seafood, in Quality Assurance in the Fish Industry: Proceedings of an International Conference, Copenhagen, August 1991 Eds Huss

H.H., Jakobsen M., Liston J Amsterdam, Elsevier 1992, 93-105.

Gram L Evaluation of the bacteriological quality of seafood International Journal of Food Microbiology, 1992, 16 (1), 25-39.

Hobbs G Fish: microbiological spoilage and safety Food Science and Technology Today,

Pedrosa-Menabrito A., Regenstein J.M Shelf-life extension of fresh fish - a review Part I

- spoilage of fish Journal of Food Quality, 1988, 11 (2), 117-27.

Hackney C.R., Dicharry A Seafood-borne bacterial pathogens of marine origin Food Technology, 1988, 42 (3), 104-9.

Garrett E.S Microbiological standards, guidelines, and specifications and inspection of

seafood products Food Technology, 1988, 42 (3), 90-3, 103.

Hobbs G Microbiology of fish, in Essays in Agricultural and Food Microbiology Ed.

Norris J.R Chichester, Wiley 1987, 199-226.

Eyles M.J Microbiological hazards associated with fishery products CSIRO Food Research Quarterly, 1986, 46 (1), 8-16.

Simmonds C.K., Lamprecht E.C Microbiology of frozen fish and related products, in

Microbiology of Frozen Foods Ed Robinson R.K London, Elsevier Applied

Science Publishers 1985, 169-208.

Hobbs G., Hodgkiss W The bacteriology of fish handling and processing, in

Developments in Food Microbiology, Volume 1 Ed Davies R London, Applied

Science Publishers 1982, 71-117.

1 CHILLED AND FROZEN RAW FISH

Associate Prof Covadonga Arias

Department of Fisheries and Allied Aquacultures

be used for fresh and seawater finfish

Fish are an important part of a healthy diet since they contain high qualityprotein, but typically present a low fat percent when compared to other meats Inaddition, most fish contain omega 3-fatty acids and other essential nutrients.Although fish is broadly similar in composition and structure to meat there are

a number of distinctive features Protein content in fish fillet varies typically from

16 - 21% The lipid content, which can be up to 67%, typically fluctuates between0.2 - 20%, and is mostly interspersed between the muscle fibres Fish fillets are apoor source of carbohydrates, offering less than 0.5% (1) Fish fillet compositioncan vary significantly within the same species due to feed intake, migratorypatterns, and spawning season The lipid fraction is the component showing thegreatest variation; it shows a typical season pattern especially in migratory speciessuch as herring or mackerel Fish can be divided into fatty and lean fish; lean fishare those fish that store most of their fat in the liver, while fatty fish have fat cellsdistributed along their bodies Muscle composition and structure of fish also differfrom those found in other meat Fish flesh is dominated by the abundance of whitemuscle in relatively short segments, giving it its characteristically flaky structure.The connective tissue content of fish is also lower than that found in meat,typically 3 and 15% of total weight, respectively (1)

Chilled fish is fish that has been cooled to, and maintained at or below 7 °C, but

not below 3 °C during storage, transportation and sale

Controlled-atmosphere packaging (CAP) refers to packaging in an atmosphere

where the composition of the gases is continuously controlled during storage Thistechnique is primarily used for bulk storage

DMA is dimethyl amine.

Evisceration is the removal of the viscera from a fish.

Fresh fish is raw fish that has not been processed, frozen or preserved.

Frozen fish is fish that has been cooled to, and maintained at or below -2 °C

(normally below -12 °C) during storage, transportation and sale

Modified-atmosphere packaging (MAP) refers to packaging systems in which the

natural gaseous environment around the product is intentionally replaced by othergases, usually carbon dioxide (CO2), nitrogen (N2) and oxygen (O2) Theproportion of each component is fixed when the mixture is introduced, but nofurther control is exercised during storage

Organoleptic refers to qualities such as appearance, colour, odour and texture.

Quality refers to palatability and organoleptic characteristics such as tenderness,

juiciness, and flavour based on the maturity, marbling, colour, firmness, andtexture of the fish

Raw fish refers to fish that has not been cooked but excludes fish treated with

curing salts and/or subjected to fermentation

Shelf life is defined as the time of storage before microbial spoilage of a fish is

evident

Spoilage describes changes that render fish objectionable to consumers; hence,

spoilage microflora describes an association of microorganisms that, through theirdevelopment on fish, renders that fish objectionable to consumers

Spoilage potential is a measure of the propensity of microorganisms to render fish

objectionable to consumers through the production of offensive metabolic products

by-Superchilled fish is fish that has been cooled to, and maintained at temperatures

just below the freezing point, at -2 to -4 °C, during storage, transportation andsale

CHILLED AND FROZEN RAW FISH

Vacuum packaging (VP) refers to packaging systems in which the air is evacuated

and the package sealed

The subsurface flesh of live, healthy fish is considered sterile and should notpresent any bacteria or other microorganisms On the contrary, as with othervertebrates, microorganisms colonise the skin, gills and the gastrointestinal tract

of fish The number and diversity of microbes associated with fish depend on thegeographical location, the season and the method of harvest In general, thenatural fish microflora tends to reflect the microbial communities of thesurrounding waters It is difficult to estimate how many microorganisms aretypically associated with fish, since they heavily depend on the type of sampleanalysed and the protocol used for isolation In fact, standard culture-dependentmethods can only recover between 1 to 10% of total bacteria present in any givensample More accurate, molecular-based methods have not yet been used toaddress this issue Gastrointestinal tracts and gills typically yield high bacterianumbers, although these are influenced by water quality and feed Fish harvestedfrom clean and cold waters will present lower bacterial numbers than fish fromeutrophic and/or warm waters However, potential human pathogens may bepresent in both scenarios

The autochthonous bacterial flora of fish is dominated by Gram-negative

genera including: Acinetobacter, Flavobacterium, Moraxella, Shewanella and Pseudomonas Members of the families Vibrionaceae (Vibrio and

Photobacterium) and the Aeromonadaceae (Aeromonas spp.) are also common

aquatic bacteria, and typical of the fish flora Gram-positive organisms such as

Bacillus, Micrococcus, Clostridium, Lactobacillus and coryneforms can also be

found in varying proportions (1) It is crucial to mimic the environmental chemical parameters when isolating bacteria from fish For example, some species

physico-(most Vibrios) require sodium chloride for growth; whenever possible, several

culture media containing sodium chloride and more than one incubationtemperature should be used It must be noted that mesophilic bacteria can rapidlyovergrow psychrotropic organisms

Human pathogenic bacteria can be part of the initial microflora of fish, posing

a concern for seafoodborne illnesses (2) These pathogens can be divided into twogroups: organisms naturally present on fish (Table 1.I); and those that although notautochthonous to the aquatic environment, are present there as result ofcontamination (anthropomorphic origin or other) or are introduced to the fishduring harvest, processing or storage (Table 1.II) (3)

TABLE 1.I Indigenous bacterial pathogens typically present in fish (3) Organism Temperature range Estimated Minimum Infective

Dose

Clostridium botulinum - 3 - 26 °C 00.1 - 1 µg toxin lethal dose non-proteolytic type E

Pathogenic Vibrio spp. 10 - 37 °C High for most species,

Vibrio parahaemolyticus (exception: V vulnificus) Vibrio vulnificus

other vibrios

Plesiomonas shigelloides 8 - 37 °C Unknown

TABLE 1.II Non-indigenous bacterial pathogens frequently present in fish (3) Organism Primary habitat Minimum Infective Dose

Listeria monocytogenes* Soil, birds, sewage, Variable depending on the

stream water, estuarine strain (>10 2 cells/g) environments, and mud

Staphylococcus aureus Ubiquitous, human origin 10 5 - 10 6 cells/g Toxin

levels 0.14 - 0.19 µg/kg bodyweight

Salmonella spp. Intestinal track of from < 10 2 - >10 6

terrestrial vertebrates

Escherichia coli Fecal contamination 10 1 - 10 3

Yersinia enterocolitica Ubiquitous in environment High (10 7 - 10 9 cells/g)

* Some sources considered L monocytogenes as part of the natural aquatic flora

It is apparent from the above that there is potentially a very diverse range oforganisms present on fish However, numbers of pathogenic bacteria in raw fishtend to be low, and risk associated with the consumption of seafood is low (2, 4)

In addition, during storage indigenous spoilage bacteria tend to outgrow potentialpathogenic bacteria

Shelf life depends on the initial microflora on the fish, potential contaminantsadded during handling and processing, and conditions of storage

1.3.1 Capture, handling and processing

Wild finfish are usually caught by net, hook and line, or traps, with very littlecontrol over the condition of the fish at the time of death or the duration of thekilling process This contrasts greatly with the meat industry, in which the health

of each animal can be assessed prior to slaughter, and the killing process isdesigned to minimise stress However, in recent decades, aquaculture practiceshave been expanding worldwide, offering better control of fish health prior to, andduring harvest

The length of time that set nets have been in the water or the time trawlers’ netsare towed, has an effect on the amount of stress and physical damage that the fishwill suffer during capture Physical damage such as loss of scales, bruising andbursting of the gut will increase the number of sites open for bacterial attack andspread In addition, cortisone levels increase during prolonged stress and can alterthe fillet quality

After capture, the fish may be stored in the vessel for periods ranging from just

a few hours to several weeks in melting ice, chilled brine or refrigerated seawater

at -2 °C Inadequate circulation of chilled brines may result in localised anaerobicgrowth of some microorganisms, and spoilage, with the production of off-odours.Used refrigerated brines can be contaminated with high numbers ofpsychrotrophic spoilage bacteria, and their re-use will increase the cross-contamination of other fish with such microorganisms Increasingly, andespecially when fish is stored on board for longer periods, freezing facilities(-18 °C) may be used to prevent the catch from deteriorating

Fish may be eviscerated prior to storage at sea - a practice that may have bothadvantages and disadvantages The action of intestinal enzymes and activity of thegut bacteria on the flesh around the belly cavity may produce discolouration,digestion and off-flavours in uneviscerated fish In eviscerated fish, however, thecuttings provide areas of exposed flesh that are open to microbial attack Ifevisceration is carried out at sea, care should be taken in removing all the gutcontents and washing the carcass thoroughly prior to refrigerating, icing orfreezing The decision to eviscerate the catch at sea will depend greatly on the size

of the fish and the duration of storage at sea, with fish such as tuna and cod beingmore commonly eviscerated than sardines, mackerel or herring

During capture and storage, finfish will almost invariably come into contactwith nets, decks, ropes, boxes and/or baskets, human hands and clothing Thesecontacts will not only increase the bacterial cross-contamination between fishbatches but will introduce microorganisms from other sources such as humans,birds and soil Of particular concern is the use of wooden or soiled plasticcontainers for storage and unloading at the quayside, in which the bacterial load

CHILLED AND FROZEN RAW FISH

can be substantial These containers are also used for displaying the fish duringauction at the quayside, often in the absence of adequate refrigeration.

As with all foods, careful and sanitary handling during processing is required

to reduce the risk of contamination with potential human pathogens, and to limitthe loss of quality (5) Good Manufacturing Practice (GMP) and control of thesanitary conditions of the transport and processing environments are essential tolimit additional risk of disease caused by fish consumption (6, 7) Monitoring ofthe seawater for algal growth in order to limit the risk of algal toxin ingestion, and

of the quality of the water used for ice and to wash fish, cleaning of the workenvironment, use of effective detergents and disinfectants, and minimisinghandling will all reduce microbial cross-contamination

1.3.2 Modified-atmosphere packaging

A natural atmosphere rich in oxygen (21%) is responsible for oxidative processesand for all aerobic respiratory life Low oxygen levels have been shown tosubstantially prolong the freshness and quality life of refrigerated seafoodproducts MAP extends the shelf life of most fishery products by inhibitingbacterial growth and autoxidation

In MAP, the natural atmosphere is replaced with a controlled gas mixture(carbon dioxide, nitrogen, oxygen etc.) Carbon dioxide is the most important gas

in MAP of fish because of its bacteriostatic and fungistatic properties In theabsence of oxygen, partial fermentation of sugars occur leading to lower pH Bothcarbon dioxide and low pH inhibit the growth of the typical spoilage bacteria such

as Pseudomonas and Shewanella Bacterial composition under MAP shifts from

mostly Gram-negative to predominantly Gram-positive (lactic) bacteria

Brochothrix thermosphacta and psychrotrophic lactic acid bacteria (LAB) can

produce spoilage characteristics; however, they are usually process contaminants,not part of the normal flora of the meat animals (8) Anaerobic atmospheres haveless effect on fresh fish shelf life; fish have a higher post mortem pH, and specificspoilage organisms may use other terminal electron acceptors naturally present inthe fish (trimethylamine-N-oxide (TMAO), ferric ion (Fe3+)) What is more,potential spoilage bacteria are among the psychrophilic and psychrotrophic florapresent on temperate-water fish before death (9)

Packaging changes the intrinsic and extrinsic parameters affecting a product,from water activity (aw) through to physical damage These changes can bedeleterious, allowing more growth of spoilage organisms, for example, but ifapplied properly should extend the life of the product Physical barriers not onlyprotect from physical damage, but isolate the food in an environment differentfrom the bulk atmosphere

The atmospheric conditions surrounding a product may be passively or activelyaltered By vacuum packing foods, a reduction in the oxygen tension is achievedwhich, in time, if there is some oxygen demand from the product, will result infully anoxic conditions However, by actively altering the composition of the

CHILLED AND FROZEN RAW FISH

surrounding gas, a modified-atmosphere may contain any gas necessary for thedesired effect

Modified-atmosphere preservation of fish was first reported in the 1930s, butonly in recent years has it seen a marked expansion in use and market share Thishas been driven partly by increased consumer demand for fresh and chilledconvenience foods containing fewer chemical preservatives MAP has beenapplied to fresh meat and fish with a resulting commercially viable extension inshelf life (10) The microflora of meat is not the same as that of whole, gutted orfilleted fish, and the MAP of fish has more challenges to overcome as a result of:

a comparatively large initial load of bacteria present, which are able to growrapidly at low temperature; the higher pH and reduction potential (Eh) of the fishmuscle; the pathogens that may be able to grow before spoilage occurs; and theproblems of muscle structure damage by the modified-atmosphere (11)

VP is one of the oldest forms of altering the interior gaseous environment of apack, but residual oxygen and other electron acceptors may be sufficient to allowoxidative spoilage of fish (12)

The principal effect of raised carbon dioxide-MAP is an extension of the 'lag'phase of the growth of the bacteria on the fish, the inhibition of common 'spoilage'

bacteria (Pseudomonas, Flavobacterium, Micrococcus and Moraxella), and the

promotion of a predominantly Gram-positive, slower-growing flora (11).Many additives have been tried in conjunction with changed atmosphere,including salt, phosphates, sorbates and chelating agents such asethylenediaminetetraacetic acid (EDTA) (11) The products, after additiveapplication, may not be considered ‘fresh’ fish

Gases used in MAP of fish most commonly include carbon dioxide andnitrogen High concentrations of carbon dioxide have the most pronouncedmicrobial effect, but can dissolve into fish liquids and deform packages, discolourpigmented fish (11), and increase in-pack drip (12) Replacement of oxygen withnitrogen, an inert and odourless gas, does inhibit some aerobic bacteria and reducethe rate of oxidative rancidity Sulphur dioxide, nitrous oxide and carbonmonoxide have also been suggested as possible replacement gases in traceamounts for MAP/CAP, although less information on their effectiveness isavailable

The single most important concern with respect to the use of MAP is the

potential for outgrowth and toxin production by C botulinum Of particular

concern are the psychrotrophic type E and non-proteolytic type B and F strains, asthey are able to grow at temperatures as low as 3.3 °C and produce toxins, withoutovert signs of spoilage Growth and toxin production have been detected inartificially contaminated packs of whole trout after 1 week’s incubation at 10 °C(13)

The (International) Codex Committee has published a Code of Practice for Fishand Fishery Products (CAC/RCP 52-2003), which provides guidance relating tovacuum or modified-atmosphere packaging for specific fish products (14)

In the United States, the National Advisory Committee on MicrobiologicalCriteria for Foods (NACMCF) has published a number of recommendations on

the safety of MAP and VP refrigerated raw fishery products (15) It highlightstemperature control as the primary preventive measure against the possible hazard

of toxin production by C botulinum, leading to the recommendation that the sale

of MAP/VP raw fishery products be allowed only when certain conditions aremet These conditions include storage of the product at ≤ 3.3 °C at all points frompackaging onwards, use of high-quality raw fish, adequate product labelling inrespect of storage temperature, adequate shelf life and cooking requirements, andthe use of a HACCP plan It was also noted that organoleptic spoilage andrejection by the consumer should occur before the possibility of toxin production,

at all times

In the United Kingdom, the Sea Fish Industry Authority has publishedguidelines for the handling of fish packaged in a controlled-atmosphere (16).The 1985 British Guidelines on MAP/CAP fish from the National FisheriesInstitute state that:

1 Only the highest-quality fish, from both a microbiological and a chemicalstandpoint, should be used

2 The only processing dip to be used is 5% potassium sorbate and 10% sodiumtripolyphosphate

3 The replacement gas should contain at least 40% carbon dioxide

4 Subsequent storage must be at or below 3 °C

5 Sensitive and accurate time-temperature indicators must be used to ensurethat the product has not been temperature-abused

Although all fish species need to be treated separately, grouping of fish types

is possible to estimate the best carbon dioxide level to be used in MAP For fattyfish, oxidative rancidity is a significant source of spoilage and hence the completeremoval of oxygen and the use of oxygen-impermeable films are preferred Theaddition of high levels of carbon dioxide to these fish, however, may result inunacceptable colour and textural change and hence little shelf life extension (11).For other less fatty marine fish, however, greater advantages are seen

Storage studies have shown that the extension in shelf life of MAP cod filletswas proportional to carbon dioxide concentration up to around 50% (V/V), fromwhere drip loss and gaping of the muscle meant that organoleptically classifiedrejection occurred sooner than with no carbon dioxide addition (12) The problem

of water loss in MAP fish is common, owing to the dissolution of cellularstructures; this problem occurs to a lesser extent with meat, but is recognised as adrawback of carbon dioxide application Another problem facing the application

of MAP to marine fish is the existence of carbon dioxide-resistant bacteria that canrapidly produce off-odours (e.g trimethylamine and sulfides) The growth of thesebacteria is reduced by high carbon dioxide concentrations but, as already stated,rejection due to alteration in fish texture with greater carbon dioxide makes thisunsuitable (13)

Freshwater fish have different intrinsic bacterial flora and also do not containsuch high concentrations of amines (e.g TMAO) that can be reduced to produce

CHILLED AND FROZEN RAW FISH

off-odours, so lower carbon dioxide concentrations may have better preservativeeffects

Gas mixtures of 40% CO2/30% N2/30% O2for white fish and 40 - 60% carbondioxide with a balance of nitrogen for fatty fish have been recommended (16, 17,18) and are probably the most widely used

1.4.1 Fresh fish spoilage and methods of evaluation

Food spoilage can be considered as any change that renders the productunacceptable for human consumption (10) Spoilage of fish starts upon death due

to autoxidation (oxidation of unsaturated lipids), reactions caused by activities ofthe fish’s own enzymes, and metabolic activities of microorganisms present in thefish Over time, loss of the fresh characteristics may be simply measured bycomparative visual and smell analysis

Loss of freshness and spoilage cannot be separated as processes, but it is acommonly held view that loss of freshness is related to autolytic degradation andspoilage is more microbial in origin (1)

Degradation of whole fresh fish stored in ice generally follows a set pattern,and this pattern is the basis of freshness grading schemes The eyes turn fromconvex and clear to concave and opaque, the gills from pink and shiny, with nosmell, to brown and slimy with an intense off-odour; the skin turns from iridescent

to dull and bleached with bacterial slime; and the flesh turns from bright andelastic to dull and soft (1)

Various methods for whole fish freshness evaluation by a trained panel havebeen proposed; these are summarised in Table 1.III

TABLE 1.III Fish-quality scales

Quality Index Method 0 to 24 (arbitrary reject) European Grading Scheme Extra, A, B, C (C=reject)Significantly, rejection of whole fresh fish for human consumption may bemade without chemical or microbiological evaluation, or for that matterevaluation of the taste of the fish, as traditional spoilage patterns of the externalorgans are typically very distinct

When working with fillets, evaluation of taste is unavoidable as the visualpattern of degradation of the eyes and gills is unavailable for analysis Taste panelevaluation of cooked fish may be used to score or grade colour, texture, smell and

taste of cooked fish Generally, the cooked analysis of fish passes through fourphases of spoilage:

i) Delicate sweet, sea-weedy taste, possibly slightly metallic

ii) Neutral taste, little flavour

iii) Traces of sour, fruity and/or bitter off-flavours; development of sickly sweet,cabbage-like, ammoniacal, sulphurous and/or rancid smells; texture becomessoft and watery or hard and dry

iv) Enhancement of the spoilage characteristic of phase iii, spoiled and putrid

The processes of degradation being analysed by the quality scoring methodsabove are a complex mix of physical, chemical, biochemical and microbiologicalactions These processes are strongly influenced by the physical conditions ofstorage

After death, rigor mortis is the first noticeable change in the fish From beingflaccid, the muscles harden as residual adenosine triphosphate (ATP) is reducedand the myosin and actin filaments bind to form actomyosin (18) After sometime, rigor resolves, the muscles relax again and the fish returns to a flaccid state.The pH of the muscle will drop after death depending upon the amount ofresidual glucose or glycogen that is reduced anaerobically to lactate with the co-production of ATP; this will generally correlate with the length and severity ofrigor Because fish tend to have relatively little residual glycogen compared withmammals, the pH drop of the muscle is correspondingly less; post-rigor values aretypically in the range of pH 5.8 to 6.5 (18, 19)

After death, the Eh of fish muscle remains relatively high (20) To a varyingextent, all marine fish use/have TMAO (21), which has been ascribed a number ofpossible functions as: a trimethylamine (TMA) detoxified waste product, anosmoregulator, an anti-freeze, or simply a waste product present due tobioaccumulation (22)

TMAO permits a high Eh to remain in the muscle tissue, as little endogenousreduction of TMAO occurs (18) However, bacterial reduction of TMAO to TMA,

an intense odour compound, is significant and may even be responsible for theultimate sensory rejection of fresh cod and other fish with high initial TMAOcontent (20, 23)

Changes in the resistance of the fish skin after death are used as the basis fortests that employ an electrode measurement of skin resistance (e.g the Torrymeter,

RT Freshmeter or Fishtester) As the fish degrades, the conductance generallyincreases; thus, measurements of the falling skin resistance may be made andcompared with a calibration curve to estimate the time that the fresh fish has beenstored in ice, or its remaining shelf life (1)

The process of ATP breakdown is used as an indicator of fish freshness (24)

By measuring the concentrations of the six components, a ratio of concentration

of the hypoxanthine (Hx) and inosine (HxR) to total concentration (ATP, ADP,AMP, IMP, HxR and Hx) gives a quantity (K-value, %), which increases from 0%towards 100% with time During the initial storage, reduction in ATP and increase

CHILLED AND FROZEN RAW FISH

in hypoxanthine by endogenous enzymes allow this measurement to be used fordetermining freshness (19) However, hypoxanthine, which may also be formed

by bacteria (25), is later significantly reduced by bacterial action, so themeasurement is effective only during the initial loss of freshness This measure ishighly dependent upon the temperature of storage and may not reflect the rate ofloss of quality equally at different temperatures

Lipid oxidation and other oxidative changes lead to oxidative rancidity, colourchanges, and are especially important in the spoilage of frozen fish, as microbialspoilage is limited by the low storage temperature (1, 7) Lipid oxidation can be aresult of enzymic action or a cascade reaction initiated by free radicals (26, 27)produced by aerobic respiration and other forms of metal ion reduction (28).Oxidative rancidity is known to reduce the quality of fatty fish in particular (19,26)

Chemical degradation continues after the initial post mortem phase; however,the importance of microbial action increases with time (5, 18) Quality indicesbased upon the products of microbial metabolism do not explain changes inquality until microbial growth produces measurable changes in the fish; therefore,these measurements are usually used to quantify the amount of spoilage, not todescribe freshness

Volatile bases are the best-characterised chemical indicators of fresh fishspoilage Evaluation of Total Volatile Base-Nitrogen (TVB-N, also termed TVN),

or a specific fraction of the volatile bases, for example the TMA fraction, usingConway diffusion chambers (29) allows determination of changes of mg-N/100 gfish The Conway method (and variations of it) uses a strong inorganic base tovolatilise the bases in the fish sample, and a segregated weak acid to absorb them;the residual acid is then titrated The variation in post mortem fish pH maylikewise influence the amount of bases being liberated to the air and consequentlyaffect the odour characteristics of the fish Comparing results for different fishspecies, however, does not show correlation between muscle pH and the amount

of volatile bases contained within the fish at rejection Neither does the change in

pH during storage correlate well with the production of TVB-N

During spoilage, the majority of volatile bases are produced from the solublenon-protein nitrogen of the fish (free amino acids and other low-molecular-weightnitrogenous compounds), as significant proteolysis is observed only during thelatest stages of spoilage and after rejection (30, 31) For some fish species, acorrelation can be made between the spoilage of the fresh fish and the production

of TVB-N

The major spoilage odours and flavours of fresh fish are undoubtedlyprincipally microbial in origin, but rejection of whole fresh fish by sensorymethods such as the EC grading scheme (32) is based upon non-specific odourdetection and physical appearance

1.5 Factors Affecting Fresh Fish Spoilage

1.5.1 Temperature

By far the most effective method of reducing the rate of whole fresh fish spoilage

is temperature control (1, 5) Fish spoil as a result of the chemical, biochemicaland microbiological reactions taking place within and on the fish All chemicalreaction rate kinetics (and thereby microbial growth) are temperature-dependent;the lower the temperature of storage, the slower the spoilage processes proceed(within limits) Also, careful temperature control during storage is not onlyimportant in terms of quality loss, but also crucial for the assurance of consumersafety

The application of ice storage increases the shelf life of fresh fish from a matter

of hours at ambient temperatures to days or weeks This increase has beenreported to be moderately dependent on the temperature of the sea from which thefish are taken (33, 34) The reasons for the differences between tropical- andtemperate-water fish spoilage rates may be many, including the ability of theendogenous bacteria to grow at low temperatures and lower endogenous enzymeactivity

1.5.2 Fish-spoilage bacteria

The parameters affecting the multiplication of microorganisms in foods have beencategorised into two general groups: intrinsic (inherent qualities of the food) andextrinsic (qualities of the food environment) (35)

The factors are interactive and cannot be completely isolated For fresh fishiced immediately after capture and continually stored in ice, the results of alteringone parameter may have far-reaching consequences on others For example, byaltering the gas atmosphere (extrinsic), the pH of the fish muscle may change(intrinsic) and a different microbial population may develop due to the change inatmosphere and pH The sum of these changes may result in a different spoilageprofile

1.5.3 Recognised specific spoilage organisms (SSOs)

The degree of spoilage leading to sensory rejection of fish is partly dependent onthe perception of the consumer Not all the bacteria growing on a food will lead tothe production of objectionable characteristics; a minority are often associatedwith the majority of the spoilage The concept of specific spoilage organisms(SSOs) is not new; yoghurt spoilage by yeasts and clostridial spoilage of cheese(5) are examples where it has been recognised for many years that a particularminority of the microbial flora present in the product is responsible for itsspoilage For fresh fish, realisation that the bulk of the microbial population onnewly caught fish does not cause off-flavours and off-odours stems from work

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started in the 1940s During the 1970s, work with inoculated sterile fish blocksresulted in identification of a specific minority of microorganisms, whichproduced the characteristic spoilage compounds of the fish (30, 35, 36) Theseorganisms are described as potential spoilers, but only if or when they reachnumbers capable of producing sufficient spoilage compounds to effect rejection

do they become the SSOs of the product (9, 35)

As previously noted, the spoilage of a product may be strongly influenced bythe conditions under which the product is held; therefore, the characterisation ofspoilage of each product must be made before the identification of the responsibleagent(s) can proceed Bacteria identified as being associated with the spoilageprocess of fresh fish are as follows:

1.5.3.1 Pseudomonas spp.

The Pseudomonadaceae family represent a large and poorly defined group ofmicroorganisms They are generally characterised as Gram-negative rods, motilewith polar flagella, oxidase-positive, catalase-positive, obligate respiratorybacteria The spoilage compounds associated with the growth of psychrotrophic

Pseudomonas spp on fish are diverse and in many cases species-specific Pseudomonas spp mediated spoilage is characterised by ‘fruity’, ‘oniony’ and

‘faecal’ odours from the production of ketones, aldehydes, esters and hydrogen sulphide sulphur-containing compounds such as methyl sulphide (36,37) Members of the genus are able to produce pigments, and proteolytic andlipolytic enzymes that may affect the quality of fresh and, more especially,processed (e.g frozen) fish products

non-The spoilage of freshwater fish is generally ascribed to the growth of

Pseudomonas spp (9) and they are considered SSO of iced freshwater fish (35)

1.5.3.2 Shewanella putrefaciens

Sh putrefaciens is considered an SSO of temperate-water marine fish species

stored in ice; it is often isolated as about 1 - 10% of the total flora of fresh fishfrom temperate marine waters It is also present in fresh water, and may play somerole in the spoilage of freshwater fish It is able to grow as fast as, or faster thanthe rest of the flora of ice-stored fresh marine fish (9)

The importance of Sh putrefaciens to the spoilage of fresh marine fish has been

recognised since the 1940s, although only since the late 1960s has it been realised

that specific metabolites of Sh putrefaciens growth may be used as indicators of spoilage (30) Sh putrefaciens spoilage of fish is due to its biochemical action on

muscle, i.e its ability to reduce TMAO to TMA, produce hydrogen sulphide (H2S)from cysteine, form methylmercaptane (CH3SH) and dimethylsuphide ((CH3)2S)from methionine and produce hypoxanthine (Hx) from inosine monophosphate(IMP) or inosine, plus other characteristic compounds of the species responsiblefor spoilage It is thus an important spoilage organism of gadoid fish such as cod,

for which the most compelling evidence showing spoilage as a result of Sh putrefaciens growth has been collected (12, 38) It is also able to produce

hydrogen sulphide and a range of other off-odour compounds

In terms of its taxonomy, Sh putrefaciens strains are characterised as

microaerophiles or anaerobes, they are heterogeneous, and recent taxonomicdescription using modern molecular methods bifurcated this species, with several

new species being described Initially, mesophilic strains of Sh putrefaciens were identified as Shewanella algae, Shewanella waksmanii, Shewanella affinis, and Shewanella aquimarina Later, additional psychrotrophic species such as Shewanalla baltica, Shewanella oneidensis, Shewanella gelidimarina, Shewanella frigidimarina, Shewanella livingstonensis, Shewanella olleyana, Shewanella denitrificans, and Shewanella profunda were described In fact, Sh baltica has been identified as the main H2S producer in cod during cold storing(37)

Sh putrefaciens may grow in the absence of oxygen using alternative terminal

electron acceptors (ATECs), although, like all members of thePseudomonadaceae, it is strictly respiratory This wide-ranging respiratorycapability is thought to be unique (39) The prevalence of the bacterium in somany environments, and its metal ion reduction ability has led to intensiveinvestigation into its role in iron and sulphur cycles

The ferric reductase activity of Sh putrefaciens has been studied; as the ferric

iron is insoluble, the bacteria have a significant problem to overcome - how toreduce a molecule that they are not able to take into their cells efficiently Thelocalisation of the reductase on the outer cellular membrane offers the bacteria a

solution to this problem (40) Another distinct feature of the physiology of Sh putrefaciens lies in its chemotaxis: the bacterium, unlike many other motile

bacteria, does not show carbon-source chemotaxis, but does show strongchemotaxis up gradients of most of their alternative electron acceptors (39)

1.5.3.3 Photobacterium phosphoreum

Recognised for some time as being present on spoiling fish (30), Ph phosphoreum

increased in notoriety when it was proposed that reduction of TMAO to TMAlimited the shelf life of MAP cod fillets (12) Because no other TMAO-reducingbacteria were present in sufficient numbers to produce the quantities of TMA thatwere related to rejection, it was proposed that this organism, owing to its cell sizeand activity, was capable of being in a significant numerical minority on the MAPfish but still able to yield the majority of the TMA thought to be responsible forthe rejection (23, 41) Research has shown that approximately 107 cfu/g of Ph phosphoreum were required for 50% of taste panellists to reject a sample, whereas

>108cfu/g Sh putrefaciens were required In 50% N2/50% CO2MAP cod, at the

time of rejection, a population of Ph phosphoreum sufficient to cause spoilage was found, but Sh putrefaciens was not present in such numbers (23, 41).

Ph phosphoreum has also been identified as responsible for histamine fish

poisoning This type of intoxication occurs when bacteria convert the histidine

CHILLED AND FROZEN RAW FISH

present in fish into histamine Some Ph phosphoreum strains have great capacity

as histamine producers even under refrigeration conditions

1.5.3.4 Brochothrix thermosphacta and lactic acid bacteria

B thermosphacta is a well-characterised psychrophilic spoilage organism of meat When inoculated into VP corned beef and sliced ham, B thermosphacta did not

produce off-flavours until 2 - 3 days after having reached 108cfu/g (42)

Growing evidence suggests a role for B thermosphacta in the spoilage of some MAP fish Recent studies have investigated the dominance of B thermosphacta

on spoiling fish in a 40% CO2/30% N2/30% O2MAP (42) Acetate production hasbeen reported as a good indicator of spoilage by this organism

MAP studies have also demonstrated this organism’s sensitivity to oxygen andhave shown that they are also inhibited by high CO2concentration (43)

Endogenous chemicals, algal toxins, human viruses, bacteria and higher parasitesall present some risk associated with fish consumption (1, 5, 6) Of these, it isbacterial risks that increase after capture of the fish

There are few human bacterial pathogens that can cause primary infections ordisease and are capable of persisting in the aquatic environment Fewer still arecapable of growing on fresh fish The remaining few bacteria present a major riskinvolved with the consumption of raw seafood such as sushi or oysters, but withproper cooking, these risks are substantially reduced (1, 5)

1.6.1 Clostridium botulinum

C botulinum, a convenient but diverse species of bacterium with a number of

different types, presents a potential risk The heterogeneity of the types (A, B, C1,C2, D, E, F and G) is well documented (44) Types A, B, E, (and very rarely F andG) have been reported to cause human disease; types B, E and F include bacteriawith minimum growth temperatures of approximately 3.3 °C (Group II) and theseare non-proteolytic (i.e they do not produce significant product spoilage) Adultdisease is caused by the production of the botulinum toxin, a neurotoxin thatcauses flaccid paralysis and is associated with a variable (44) mortality dependingupon dose, age, previous exposure and access to supportive treatment, includingantisera

Review of outbreak data suggests fresh fish to be safe Huss (1, 5) reports thatfresh fish consumption has never been shown to cause human botulism; this isprobably due to spoilage occurring before toxin elaboration However, lightlypreserved fish products are associated with botulism, especially type E botulinum

intoxication Of 404 intoxication outbreaks of type E C botulinum recorded up

to 1963, 75% (with a 35% mortality rate) occurred in Japan, where a particular

fermented raw fish product that is consumed uncooked, I-sushi, accounted for themajority of cases (1, 5) Other lightly or semi-preserved (e.g smoked, salted orpickled) fish have also been associated with botulism; again, the majority areassociated with products not cooked immediately before consumption The risk oftoxin formation before apparent spoilage has been studied for some MAP fish(45), but as fresh fish are often cooked before consumption and the botulinumtoxin is heat-labile, there remains only an ‘extremely small’ risk of intoxication ifreasonable precautions are taken during handling, storage and preparation (11).

Despite the ubiquitous nature of type E C botulinum in the marine

environment (isolated from around 90% of marine and environmental samplesfrom northern European waters and its consequent presence in seafood (asurveyed incidence of as high as 65%) (2, 11), the risk posed to the consumer offresh fish, whether stored aerobically or in modified-atmospheres, is small Propertemperature control (<3.3 °C) will eliminate all risk, and proper cooking (boilingfor 1 minute, or cooking at 80 °C for 5 minutes) (44) will substantially reduce risk

1.6.2 Vibrio parahaemolyticus and other vibrios

V parahaemolyticus is a marine organism, which can cause human gastroenteritis.

It is generally undetectable in marine water below 19 °C but may grow in culture

at temperatures as low as 5 °C and on food at 10 °C Only about 1% of marineisolates produce a thermostable haemolysin, which is believed to be required forvirulence Generally, only shellfish are associated with the disease and no reportedcases by the Center for Diseases Control, USA, were associated with finfishbetween 1978 and 1998 (46) The first report of the organism being a foodborneagent was with shirasu, a Japanese boiled and semi-dried sardine dish, which wasprobably contaminated from an uncooked food source or an excreting food-handler In Japan, the majority of outbreaks are caused by consumption of raw fishproducts (e.g sushi) (5), due to the cultural preference for raw fish dishes

Since 1996, V parahaemolyticus cases have increased across the world A unique clone of V parahaemolyticus O3:K6 is responsible for many of the recent

V parahaemolyticus outbreaks, including epidemics in India, France, Russia,

Southeast Asia, Japan, and North America This strain has been responsible for 50

to 80% of all V parahaemolyticus infections since 1996 and is referred to as the

pandemic strain (47) Currently, there is no specific guideline that describes a

minimum level of V parahaemolyticus in sea water fish and shellfish that could

potentially be hazardous to humans Proper chilling, use of post harvest treatmentsand/or cooking of fresh seafood will reduce risks

V vulnificus is associated with warm marine and estuarine waters Human

disease caused by the organism has only been observed in conjunction withmarine bivalve (mostly oysters) and some crustacean consumption, and when incontact with contaminated water (no CDC reported cases from fish, 1978 - 2005).The bacterium causes primary septicaemia, especially in individuals withunderlying diseases (patients suffer from immune and liver diseases or blood

CHILLED AND FROZEN RAW FISH

disorders), which is often fatal (>50%) There is no risk associated with theconsumption of properly chilled and cooked fresh fish

V cholerae O1, the causative agent of cholera, is historically associated with

faecally contaminated water, but the bacterium is known to survive and grow inthe shallow marine, and especially estuarine environment It is particularlyassociated with disease following consumption of raw oysters from warm sewage-polluted waters Again, there is no risk from the consumption of properly handledand cooked fresh fish

1.6.3 Aeromonas

Aeromonas spp., and especially Aeromonas hydrophila, are associated with

human diarrhoeal illnesses They are aquatic organisms with an epidemiology that

is yet to be fully understood The major virulence factor appears to be theproduction of toxins with enterotoxic, cytotoxic, sodium channel blocking andhaemolytic activity (48), although lack of data concerning infectious doses and thepossibility that foodborne virulence is linked to the immune state of the host meanthat full risk assessment is impossible Some isolates are true psychrophiles, withminimum growth temperatures around 0 °C and optima of 15 - 20 °C; others arepsychrotrophic mesophiles (49) Although the psychrophiles are unable to grow atbody temperature, their potential to produce disease-causing exotoxin in food hasnot been fully examined (48)

It is known that Aeromonas spp are present on fish and can grow to significant

numbers during storage (1, 5, 17) Despite this, no more than circumstantial

evidence exists linking the consumption of seafood to Aeromonas spp infection, possibly due to under-reporting because of their likeness to E coli on isolation

media (50) Most cases have been sporadic rather than associated to largeoutbreaks Chill storage may not eliminate growth, but proper cooking shouldsubstantially reduce risk of infection

P shigelloides is similar to Aeromonas spp in its habitat, though it is a true

mesophile with a minimum growth temperature of 8 °C and shows consequentialseasonal variation in its environmental isolation The bacterium has beendocumented as being responsible for a few fishborne outbreaks of gastroenteritis,and fish and shellfish are probably the major reservoirs for the organism (50)

Similar problems as those encountered for Aeromonas spp in assessing the risk of fishborne infection by P shigelloides are encountered; however, proper chill

storage will eliminate any risk associated with this organism, and proper cookingwill substantially reduce risk

1.6.4 Listeria monocytogenes

L monocytogenes has been well documented as a foodborne human pathogen Since 2000, listeriosis cases have been reportable to the CDC In the US, L monocytogenes incidence has been between 0.26 to 0.55 cases per 100,000

persons It is environmentally ubiquitous but its true frequency in the marineenvironment is not well studied It is regularly isolated from seafoods (51) Being

a psychrotroph, there is a possibility for growth of the organism on chill-storedfish, but cooking will significantly reduce the risk of infection Owing to thedependence of the virulence of this organism on the immune state of the host(usually requiring lowered cellular immunity; at risk groups - pregnant women,foetuses, neonates, alcoholics, AIDS patients and patients undergoingimmunosuppressive therapy) and a lack of data covering infectious doses, full riskanalysis is not possible (52) The organism poses a serious risk in chilled productsnot cooked before consumption, but proper cooking will reduce any risk D60of1.98 minutes in cod and D60of 4.48 minutes in salmon have been reported (53)

1.6.5 Scombroid fish poisoning

The production of histamine and other biogenic amines with humanimmunological activity, within high histidine-containing fish (especially members

of the Scombridae and Scomberesocidae families) is responsible for anintoxication termed scombroid poisoning (54) Like other toxins, it is not apparent

to the consumer and it cannot be destroyed by cooking Fortunately, scombroidpoisoning is usually a mild intoxication and it is not a significant cause of death

Of fish-transmitted human diseases scombroid poisoning is commonlyreported in the US (118 outbreaks with 463 cases reported by CDC 1998 - 2002(55)) and is also common worldwide Decarboxylation of histidine by a wide

range of bacteria including Enterobacteriacae, some Vibrio spp., Photobacterium spp., Clostridium spp and Lactobacillus spp., but more especially by Morganella morganii, Klebsiella pneumoniae and Hafnia alvei, leads to the production of

histamine, which has a maximum permissible level (for fish products from fishspecies associated with high amount of histidine) of 200 mg/kg fish in the EC Iffish are not properly refrigerated, there is a far greater risk of unacceptablehistamine levels being attained before sensory rejection Lightly preservedproducts, especially pickled fish, are exceedingly difficult to produce within thelegal limits of histamine as the temperatures used during production can lead torapid growth of histidine decarboxylating bacteria Low-temperature storage ofpotentially toxigenic (<5 °C) fish at all times is the most effective way to controlhistamine production (6)

1.6.6 Parasites

Helminthic parasites can occur extensively in finfish but very few are capable ofinfecting humans The most frequently reported parasites of human importance in

fish are round worms of the genera Anisakis and Pseudoterranova and tapeworms

of the genus Diphyllobothrium Larvae of parasites such as Anisakis simplex are

resistant to curing and marinating but can be easily destroyed by freezing at -17 to-20 °C for 2 hrs Infections are mostly associated with the consumption of raw or

CHILLED AND FROZEN RAW FISH

mildly processed fish such as sushi A limited number of nematodes and trematodesfound in finfish have also been identified as a cause of human disease (56)

In summary, risk of human disease caused by natural bacterial contamination(not through faecal pollution) of fresh fish is extremely low The most effectivecontrol of all bacterial risks is continual chill storage in ice (0 °C) or freezing,which in many cases will eliminate the risk Cooking properly immediately beforeconsumption is also an effective way of reducing or eliminating risk of freshfishborne disease

Microbiological criteria for food defines the acceptability of a product or a foodlot based on the absence or presence or number of microorganisms, includingparasites and/or quantity of their toxins / metabolites per unit of mass, volume,area or lot (CAC,1997; EC, 1997) Huss H.H., Ababouch L., Gram L Assessmentand management of seafood safety and quality FAO Fisheries Technical Paper

No 444 Rome, FAO 2003

Criteria should set standards that are attainable by the currently accepted GMPand applied only when there is absolute need for it Testing methods should bepractical and the enforcement of such criteria should translate as a reduction ofpotential microbiological risks to consumers

The most widely accepted microbiological criteria for chilled and frozen raw

fish are those set for aerobic plate counts (APC) at 25 °C and E coli proposed by

the International Commission on Microbiological Specifications for Foods(ICMSF) An increase of APC to levels in excess of 106cfu/g is usually indicative

of inadequate refrigeration, long storage under refrigeration or one of the former

prior to freezing Faecal coliform counts may be used instead of E coli counts

where this method is preferred For fish from inshore or inland waters of doubtfulmicrobiological quality, especially in warm-water areas and where fish are to be

consumed raw, it may be desirable to test for Salmonella and V parahaemolyticus

(see Table 1.IV) (57)

TABLE1.IV Sampling plans and recommended microbiological limits for fresh and frozen fish

(Adapted from ICMSF 1986)

Limit per gram or cm 2

1.8 References

1. Huss H.H Quality and quality changes in fresh fish, in FAO Fisheries Technical Paper No 348 Ed Food and Agriculture Organisation Rome, Food and Agriculture

Organisation of the United Nations 1995.

2 Davies A.R., Capell C., Jehanno D., Nychas G.J.E., Kirby R M Incidence of

foodborne pathogens in European fish Food Control, 2001, 12, 67-71.

3. Huss H.H Control of indigenous pathogenic bacterial in seafood Food Control,

1997, 8 (2), 91-8.

4. Feldhusen F The role of seafood in bacterial foodborne diseases Microbes and Infection, 2000, 2, 1651-60.

5. Huss H.H Assurance of seafood quality, in FAO Fisheries Technical Paper No.

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Organisation of the United Nations 1994.

6 Ahmed F.E Review: Assessing and managing risk due to consumption of seafood

contaminated with microorganisms, parasites, and natural toxins International Journal of Food Microbiology, 1992, 27, 243-60.

7 Ashie I.N.A., Smith J.P., Simpson B.K Spoilage and shelf life extension of fresh

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8 Davey K.R Theoretical analysis of two hot water cabinet systems for

decontamination of sides of beef International Journal of Food Science and Technology, 1989, 24, 291-304.

9 Gram L., Huss H.H Microbiological spoilage of fish and fish products.

International Journal of Food Microbiology, 1996, 33, 121-37.

10 Sivertsvik M., Jeksrud W.K., Rosnes J.T A review of modified atmosphere packaging of fish and fishery products – significance of microbial growth,

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11 Reddy N.R., Armstrong D.J., Rhodehamel E.J., Kautter D.A Shelf life extension and safety concerns about fresh fishery products packaged under modified

atmospheres: a review Journal of Food Safety, 1992, 12, 87-118.

12 Dalgaard P., Gram L., Huss H.H Spoilage and shelf life of cod fillets packed in

vacuum or modified atmospheres International Journal of Food Microbiology,

1993, 19, 283-94.

13 Gibbs P.A., Davies A.R., Fletcher R.S Incidence and growth of psychrotrophic

Clostridium botulinum in foods Food Control, 1994, 5 (1), 5-7.

14. Codex Alimentarius Commission Code of Practice for Fish and Fishery Products (CAC/RCP 52-2003) Italy, Codex Alimentarius Commission 2003.

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15 National Advisory Committee on Microbiological Criteria for Foods Vacuum or Modified Atmosphere Packaging for Refrigerated Raw Fishery Products Adopted, March 20, 1992 Washington D.C, National Advisory Committee on

Microbiological Criteria for Foods 2007.

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Part I - spoilage of fish Journal of Food Quality, 1987, 11, 117-27.

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International Journal of Food Microbiology, 1996, 33, 121-37.

21 Hebard C.E., Flick G.J.J., Martin R.E Occurrence and significance of

trimethylamine oxide and its derivatives in fish and shellfish, in Chemistry and Biology of Marine Food Products Eds Martin R.E., Flick G.J.J., Hebard C.E.

Westport, AVI Publishing 1982.

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23 Dalgaard P Qualitative and quantitative characterisation of spoilage bacteria from

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24 Saito T., Arai K., Yajima T Changes in purine nucleotide of red lateral muscle of

rainbow trout Nature, 1959, 184, 1415.

25 Surette M.E., Gill T.A., Leblanc P.J Biochemical basis of post-mortem nucleotide

catabolism in cod (Gadus morhua) and its relationship to spoilage Journal of Agricultural and Food Chemistry, 1988, 36, 19-22.

26 Ackman R.G., Ratnayake W.M.N Non-enzymatic oxidation of seafood lipids, in

Advances in Seafood Biochemistry Eds Flick G.J., Martin R.E Lancaster,

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27 Huis in't Veld J.H.J Microbial and biochemical spoilage of foods: an overview.

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29 Conway E.J., Byrne A An absorption apparatus for the micro-determination of

certain volatile substances 1 The micro-determination of ammonia Biochemical Journal, 1933, 27, 419-29.

30 Herbert R.A., Hendrie M.S., Gibson D.M., Shewan J.M Bacteria active in the

spoilage of certain seafoods Journal of Applied Bacteriology, 1971, 34, 41-50.

31 Karnop G Role of proteolytic bacteria in fish spoilage II Occurrence and

importance of proteolytic bacteria as spoilage indicators Archiv für Lebensmittelhygiene, 1982, 33, 61-6.

32 COUNCIL REGULATION (EC) No 2406/96 of 26 November 1996 laying down common marketing standards for certain fishery products http://eur-

lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:1996R2406:20050602:E N:PDF

33 Sumner J.L., Gorczyca E., Cohen D., Brady P Do fish from tropical waters spoil

less rapidly in ice than fish from temperate waters? Food Technology in Australia,

1984, 36, 328-9, 334.

34. Sumner J., Magno-Orejana F Do Tropical Fish Keep Longer in Ice Than

Temperate Fish: The Circumstantial and Definitive Approaches Rome, FAO

Fisheries Report 1985, 317, 62-70.

35 Gram L., Dalgaard P Fish spoilage bacteria – problems and solutions.

Environmental Biotechnology, 2002, 13, 262-6.

36 Miller A., Scanlan R.A., Lee J.S., Libbey L.M Volatile compounds produced in

sterile fish muscle (Sebastes melanops) by Pseudomonas putrefaciens, Pseudomonas fluorescens, and an Achromobacter species Applied Microbiology,

1973, 26, 18-21.

37 Vogel B.F., Venkateswaran K., Satomi M., Gram L Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish

marine fish Applied and Environmental Microbiology, 2005, 71, 6689-97.

38. Jørgensen B.R., Huss H.H Growth and activity of Shewanella putrefaciens isolated from spoiling fish International Journal of Food Microbiology, 1989, 9,

51-62.

39 Nealson K.H., Saffarini D Iron and manganese in anaerobic respiration:

environmental significance, physiology, and regulation Annual Review of Microbiology, 1994, 48, 311-43.

40 Myers C.R., Myers J.M Localisation of cytochromes to the outer membrane of

anaerobically grown Shewanella putrefaciens Journal of Bacteriology, 1992, 174,

3429-38.

41. Dalgaard P The effect of storage temperature on shelf life, in Fresh Fish, Quality and Quality Changes Ed Huss H.H Rome, Food and Agricultural Organisation of

the United Nations 1995, 72-80.

42. Drosinos E.H., Nychas G.J.E Brochothrix thermosphacta, a dominant

microorganism in Mediterranean fresh fish (Sparus aurata) stored under modified atmosphere Italian Journal of Food Science, 1996, 4, 323-9.

43 Dixon N.M., Kell D.B The inhibition by CO2of the growth and metabolism of

microorganisms Journal of Applied Bacteriology, 1989, 67, 109-36.

44. Taussig M.J Processes in Pathology and Microbiology Oxford, Blackwell

Scientific Publications 1984.

CHILLED AND FROZEN RAW FISH

45 Garcia G.W., Genigeorgis C., Lindroth S Risk of growth and toxin production by

Clostridium botulinum non-proteolytic types B, E and F in salmon fillets stored under modified atmospheres at low and abused temperatures Journal of Food Protection, 1987, 50, 330-6.

46 Daniels N.A., MacKinnon L., Bishop R., Altekruse S., Ray B., Hammond R.M.,

Thompson S., Wilson S., Bean N.H., Griffin P.M., Slutsker L Vibrio parahaemolyticus Infections in the United States, 1973-1998 Journal of Infectious Diseases, 2000, 181, 1661-6.

47 Myers M.L., Panicker G., Bej A.K PCR detection of a newly emerged pandemic

Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico Applied and Environmental Microbiology, 2003, 69, 2194-

200.

48 Majeed K.N., Mac Rae I.C Experimental evidence for toxin production by

Aeromonas hydrophila and Aeromonas sobria in a meat extract at low temperatures International Journal of Food Microbiology, 1991, 12, 181-8.

49. Jay J.M., Loessner M.J., Golden D.A Modern Food Microbiology USA, Springer.

52 Miller A.J., Whiting R.C., Smith J.L Use of risk assessment to reduce Listeriosis

incidence Food Technology, 1997, 51 (4), 100-3.

53. Embarek B.P.K., Huss H.H Heat resistence of Listeria monocytogenes in vacuum packaged pasteurised fish fillets International Journal of Food Microbiology,

1993, 20, 85-95.

54. Lehane L., Olley J Histamine fish poisoning revisited International Journal of Food Microbiology, 2000, 58, 1-37.

55 Lynch M., Painter J., Woodruff R., Braden C Surveillance for Foodborne-Disease

Outbreaks - United States, 1998-2002 Morbidity and Mortality Weekly Report,

2006, 55 (SS10), 1-34.

56 The International Commission on Microbiological Specifications for Foods Fish

and Fish Products (Microorganisms in Fish Products), in Microorganisms in Foods

6 Microbial Ecology of Food Commodities Ed The International Commission on

Microbiological Specifications for Foods New York, Kluwer Academic/Plenum Publishers 2005, 172-249.

57 The International Commission on Microbiological Specifications for Foods.

Microorganisms in Foods 2 Sampling for Microbiological Analysis: Principles and Specific Application Oxford, Blackwell Scientific Publications 1986, 181-96.

1.9 Further Reading

1.9.1 Processing

Anon Handling and processing of scad, in Torry Advisory Note No 93 Aberdeen, Torry

Research Station of the Ministry of Agriculture, Fisheries and Food 1989 Labuza T.P., Taoukis P.S Prediction of shelf life and safety of minimally processed

CAP/MAP chilled foods: a review Journal of Food Protection, 1992, 55, 741-50 Cann D.C., Smith G.L., Houston N.G., Torry Research Station Further Studies on Marine Fish Stored Under Modified Atmosphere Packaging Aberdeen, Torry Research

Station of the Ministry of Agriculture, Fisheries and Food.1983.

Villemure G., Simard R.E., Picard G Bulk storage of cod fillets and gutted cod (Gadus morhua) under carbon dioxide atmosphere Journal of Food Science, 1986, 51,

317-21.

Cho Y., Shinano H., Akiba M Studies on the microbiological ecology of mackerel stored

by the method of partial freezing 1 Changes in microflora and chemical

compounds in mackerel stored by partial freezing Bulletin of the Faculty of Fisheries (Hokkaido University), 1984, 35, 271-85.

1.9.2 Spoilage

Shewan J.M The Microbiology of Seawater Fish, in Fish as Food Ed Borgstrom G.

Florida, Academic Press 1961.

McMeekin T.A., Ross T., Olley J Application of predictive microbiology to assure the

quality and safety of fish and fish products International Journal of Food Microbiology, 1992, 15, 13-32.

Gibson D.M., Ogden I.D., Hobbs G Estimation of the bacteriological quality of fish by

automated conductance measurements International Journal of Food Microbiology, 1984, 1, 127-34.

Dalgaard P., Huss H.H Mathematical Modelling Used for Evaluation and Prediction of

Microbial Fish Spoilage, in Seafood Science and Technology Eds Shahidi F.,

Jones Y., Kitts D.D Lancaster, Technomic Publishers 1997, 73-89.

Gram L., Trolle G., Huss H.H Detection of specific spoilage bacteria from fish stored at

low (0 °C) and high (20 °C) temperatures International Journal of Food Microbiology, 1989, 4, 65-72.

Sumner J., Magno-Orejana F Do Tropical Fish Keep Longer in Ice than Temperate Fish:

the Circumstantial and Definitive Approaches, in FAO Fisheries Report No 317 (Suppl.) Ed Food and Agriculture Organisation of the United Nations Rome,

Food and Agriculture Organisation 1985, 62-70.

Ravn Jørgensen B., Gibson D.M., Huss H.H Microbiological quality and shelf life

prediction of chilled fish International Journal of Food Microbiology, 1988, 6,

295-307.

CHILLED AND FROZEN RAW FISH

Einarsson H Predicting the shelf life of cod (Gadus morhua) fillets stored in air and modified atmosphere at temperatures between -4 °C and +16 °C, in Quality Assurance in the Fish Industry Eds Huss H.H., Jacobsen M., Liston J.

Amsterdam, Elsevier Science 1992, 479-88.

Gram L., Trolle G., Huss H.H Detection of specific spoilage bacteria from fish stored at

low (0 °C) and high (20 °C) temperatures International Journal of Food Microbiology, 1987, 4, 65-72.

1.9.3 Pathogens

Condon S., Garcia M.L., Otero A., Sala F.J Effect of culture age, pre-incubation at low

temperature and pH on the thermal resistance of Aeromonas hydrophila Journal of Applied Bacteriology, 1992, 72, 322-6.

Gram L Inhibitory effect against pathogenic and spoilage bacteria of Pseudomonas strains isolated from spoiled and fresh fish Applied and Environmental Microbiology, 1993, 59, 2197-203.

Klausen N.K., Huss H.H A rapid method for detection of histamine producing bacteria.

International Journal of Food Microbiology, 1987, 5, 137-46.

1.9.4 Standards and criteria

Council Regulation (EC) Number 2406/96 of 26 November 1996 laying down common marketing standards for certain fishery products.

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:1996R2406: 20050602:EN:PDF

Davies A.R Modified atmosphere packaging of fish and fish products, in Fish processing technology Ed Hall G.M London, Blackie Academic & Professional 1997, 200-

23.