detection of fish spoilage … · 29.2 types of spoilage ... spoilage is a natural phenomenon that...

537

Chapter 29

Detection of Fish Spoilage

George-John E. Nychas and E.H. Drosinos

Contents29.1 Introduction ..................................................................................................................53729.2 Types of Spoilage ...........................................................................................................538

29.2.1 Microbial Spoilage ...........................................................................................53929.2.2 Ephemeral Spoilage Organisms ........................................................................53929.2.3 Physicochemical Spoilage—Fat Oxidation ..................................................... 54429.2.4 Enzymatic Spoilage ......................................................................................... 544

29.3 Determination Methods ............................................................................................... 54429.3.1 Sensory Analysis ..............................................................................................54529.3.2 Total Volatile Bases (TVB) and Trimethylamine (TMA) Determination ........545

29.3.2.1 Solid-Phase Microextraction ............................................................54529.3.2.2 HS/MS Analysis ............................................................................. 546

29.3.3 Detection of Fish Spoilage by Colorimetry—Texture Assessment ................... 54629.3.4 Quantifying Biogenic Amines......................................................................... 54729.3.5 Microbiological Analyses ................................................................................ 54829.3.6 Rapid Methods for Detection and Quantifi cation of Microorganisms ............ 549

29.3.6.1 Short-Wavelength Near-Infrared Spectroscopic Method (SW-NIR) ....................................................................................... 549

29.3.7 Predicting Fish Spoilage .................................................................................. 54929.4 Conclusions ...................................................................................................................551References ................................................................................................................................551

29.1 IntroductionSpoilage is a natural phenomenon that leads to the decomposition of a food substratum. In particular, the spoilage of fi sh can be considered as an ecological phenomenon that encompasses

© 2010 by Taylor and Francis Group, LLC

538 ◾ Handbook of Seafood and Seafood Products Analysis

a series of changes in the available components (e.g., low molecular weight compounds) through (1) natural processes (e.g., chemical, enzymatic activity, or even autolytic process) and (2) micro-bial proliferation. A general feature of microbial spoilage is its relatively sudden onset, i.e., it does not appear to develop gradually, but more often as an unexpected and unpleasant revelation. Th is is a refl ection of the exponential nature of microbial growth and its consequence that microbial metabolism can also proceed at an exponentially increasing rate. If a microbial product associated with spoilage, for example an off odor, has a certain detection threshold, the level will be well below this threshold for most of the product’s acceptable shelf life.

Many eff orts to delay this natural process encompass certain strategies that are determined by general factors of the society. Nowadays the mild preservation of foods and the eff ort to extend the food shelf life have changed the status of the food enterprises. Quality and food safety manage-ment systems are planned, established, and applied, operated and maintained to satisfy the con-sumers’ needs, expectations, and requirements. Monitoring and validation of the control measures applied are essential parts of the applied systems. Detection of spoilage and estimation of shelf life is of paramount importance during the operation of these systems [25].

It is important to determine the nature of the ephemeral spoilage organisms (ESO) growth and dominance in fi sh and fi sh products. Based on this information it is more convenient to establish indexes that refl ect the microbiological quality of fi sh. In addition, microbiological quality will be interpreted in terms of spoilage [9,23,26,36,37,48].

Fish spoilage is a complex process in which physical, chemical, and microbiological mecha-nisms are implicated. Upon fi sh harvesting, the fi sh regulatory mechanisms, which prevent inva-sion of the tissues by bacteria, cease to function—bacteria or the enzymes invade the fl esh of fi sh. Th is process produces toxic compounds in the fi sh and the fi sh becomes spoiled. Th e initial stages of fi sh spoilage is characterized by the loss of characteristic odor and taste are mainly due to autolytic degradation, while the fi nal stages of fi sh quality deterioration is characterized by soften-ing or toughening of fl esh texture along with the production of unpleasant odors and fl avors that are mainly due to microbial activity [4,17].

Another cause of fi sh spoilage is lipid oxidation and hydrolysis that leads to the development of rancidity, even with storage at subzero temperatures. Th is is due to the large amount of polyun-saturated fatty acid moieties found in fi sh lipids. In fact, this is a major cause of frozen fi sh spoilage [28]. During the spoilage, characteristic changes in sensory attributes relate to the appearance, aroma, taste, texture, and appearance will change [51].

Since fi sh is a very perishable commodity, it has drawn special attention. As most raw materials, fi sh consist of a large number of species of widely diff ering appearance and fl avor so that customers are often unsure if particular species of products made from them are suitable and safe to eat. Th e public also becomes more demanding in respect of freshness, microbiological safety, free from pollutants, protection from damage, and convenience.

Quality control and labeling of fi sh products depend on defi ning the appropriate criteria, which may be of diff erent importance to the various parts of the supply chain in the fi sh sector. Fresh-ness of fi sh is the key factor determining quality, but also seasonal variations, catching methods, handling, processing, and storage techniques infl uence quality [18,20].

29.2 Types of SpoilageSpoilage of fi sh and seafood is caused by microbial, oxidative, and enzymatic activity which results in undesirable changes in odor, fl avor, and texture [50]. pH of fi sh muscle is an important factor


Detection of Fish Spoilage ◾ 539

of spoilage. pH values higher than 7, observed when fi sh is exhausted in glycogen during netting operations, is favorable for microbial and enzymatic spoilage. Evisceration plays an important role in spoilage, e.g., small pelagics are not gutted, hence proteolytic enzymes along with microorgan-isms rapidly invade the fi sh’s tissues. Oily fi sh are susceptible to oxidative rancidity. Cold water species of fi sh, due to their microbiota psychrophilic, are predisposed to earlier spoilage under chill conditions. Type and rate of spoilage is multifactorial [45].

29.2.1 Microbial SpoilageFish spoilage commences upon harvesting. At that time, fi sh come in contact with a totally diff er-ent microbiota. Microbial activity is retarded, delaying but not inhibiting the spoilage of fi sh dur-ing storage conditions at the temperature of 0°C. Gram-negative, fermentative bacteria (such as Vibrionaceae) spoil unpreserved fi sh, whereas psychrotolerant Gram-negative bacteria (Pseudomo-nas spp. and Shewanella spp.) grow on chilled fi sh [40].

Fish, as a poikilothermic organism, possess a microfl ora infl uenced by the temperature of the water and by the microbiota dominating the bottom sediment of the catch area. Fish caught on a line may have lower bacterial counts than fi sh that are trawled by dragging a net along the bottom; the trawl net drags through the bottom sediment, which usually has high counts of microorgan-isms. Unlike other crustacean shellfi sh (i.e., lobster, crab, and crayfi sh) that are kept alive until preparation for consumption, shrimp die soon after harvesting. Decomposition of shrimp involves bacteria on the surface that originate from the marine environment or are introduced during han-dling and washing. Molluscan shellfi sh (i.e., oysters, clams, scallops, and mussels) are sessile and fi lter feeders and, as such, their microbiota depends greatly on the quality of water in which they reside, the quality of wash water, and other factors [48].

29.2.2 Ephemeral Spoilage OrganismsStudies have established that spoilage is caused only by an ephemeral fraction of the initial microbial association [48]. Th is concept has contributed signifi cantly to our understanding of meat and seafood spoilage. Th e microbial associations developing on muscle tissues stored aero-bically at cold temperatures are characterized by an oxidative metabolism. Th e Gram-negative bacteria that spoil fi sh are either aerobes or facultative anaerobes. Pseudomonas spp., Shewanella putrefaciens, and Photobacterium phosphoreum were found to be the dominant species on fi sh muscle stored at cold temperatures (Tables 29.1 and 29.2). Brochothrix thermosphacta also occur on chilled fi sh stored mainly under modifi ed atmospheres in signifi cant numbers that con-tribute to the microbial associations [9]. Lactic acid bacteria are considered to be important in spoilage when fi sh is stored under modifi ed atmosphere packaging (MAP). Both lactic acid bacteria and B. thermosphacta are probably the most important causes of spoilage characterized by muscle souring. Th e other distinct type of muscle spoilage is characterized by putrefaction and is related to proteolytic activity and off -odor production by Gram-negative bacteria that dominate under aerobic conditions. Other common spoilage conditions and causative bacteria are listed in Table 29.3 [48,60].

In general, the metabolic activity of the ephemeral microbial association, which prevails in a muscle ecosystem leads to the manifestation of changes that are characterized as spoilage of meat. Th ese undesirable spoilage changes are related to the type, composition, and population of the microbial association and the type and availability of substrates for energy production in



fi sh (Table 29.4). Indeed the type and the extent of spoilage is governed by the availability of low-molecular-weight compounds (e.g., glucose, lactate) existing in fi sh; eventual muscle food changes and subsequent overt spoilage are due to catabolism of nitrogenous compounds as well as secondary metabolic reactions.

Table 29.1 Ephemeral Spoilage Organisms (ESOs) in Seafood

Seafood Typical ESO

Fresh and chilled, stored in air S. putrefaciens-likea,b

Pseudomonas spp.c

Fresh, chilled and stored in vacuum or modifi ed atmosphere packaging

P. phosphoreumb

Lactic acid bacteriac

B. thermosphactac

Fresh and lightly preserved products stored at ambient temperature

Aeromonas spp.

Vibrio spp.

Enterobacteriaceae

Enterococcus faecalis

Sous-vide cooked and chill stored Gram-positive spore formers

Lightly preservedd and chill stored Lactic acid bacteriae

Enterobacteriaceaef

P. phosphoreum

Vibrio spp.

Semi-preserved, salt cured and chilled Halobacterium spp., Halococcus spp., and osmotolerant molds and yeasts

Fermented and chilled Molds and lactic acid bacteria

Sources: Based on Gram L. and Huss, H.H., The Microbiological Safety and Quality of Food, Lund, B.M. et al. (Eds.), Aspen Publishers, Gaithersburg, MD, 2000, 472–502; Gram, L. and Dalgaard, P., Curr. Opin. Biotechnol., 13, 262, 2002; Lambropoulou, K., The effects of varying extrinsic parameters and specifi c pretreatments in whole fi sh and prepared fi sh fi llets, PhD Thesis, University of Lincolnshire & Humberside, Lincoln, U.K., 1999; Vogel, B.F. et al., Appl. Microbiol., 71, 6689, 2005; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140.

a Refer to S. putrefaciens, Shewanella baltica, and other closely related H2S-producing, Gram-negative bacteria.

b Typical of fi shes from marine and temperate waters.c Typical of fi shes from freshwater and fi shes from warmer waters.d Include, for example, cold-smoked salmon, cooked and brined shrimps and brined roe

products.e Include, for example, Lactobacillus curvatus and Lactobacillus sakei.



Bacteria are the major cause of spoilage of most seafood products. Th eir growth and metab-olism results in the formation of amines, sulfi des, alcohols, aldehydes, ketones, and organic acids with unpleasant and unacceptable off -fl avors [49]. Especially ephemeral spoilage organ-isms (ESOs) give rise to the off ensive off -fl avors. Although they play only a minor part of the total microbiota, they are classifi ed as active spoilers. Th ese bacteria grow in fi sh juice producing spoilage off -odors described as fi shy, rotten, or cabbage-like. Th e prominent characteristics of fi sh spoilage bacteria are an ability to reduce trimethylamine oxide (TMAO) and to produce hydrogen sulfi de (H2S). However, ESO are not the same in every case and are dependent on

Table 29.2 Main Low-Molecular-Weight Components of Beef and Fish Pre- and Post-Rigor Mortis (mg/100 g)

Component Pre Post

Creatine phosphate 9.3a 0.2a

Creatine ndb nd

Betaine nadc 100d

ATP 6.5a 0.2a

IMP nd nd

Glycogen 220 40

Glucose 220 40

Glucose-6-phosphate 21 32

Lactic acid 100 400

pH 7.3 6.5

Free amino acids nad 250

TMAO nd 350–1000

Carnosine and anserine nd 100e

Sources: Huss, H.H., Quality and Quality Changes in Fresh Fish. FAO Fisheries Technical Paper, No. 348, FAO, Rome, Italy, 1995b; Jay, J.M., Modern Food Microbiology, Aspen Publishers, Gaithersburg, MD, 2000; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140.

a μmol/g.b nd: not determined.c nad: no available data.d In some fi sh.e Cod.



the climatic and storage conditions, the type of fi sh and even the place in which the fi sh was harvested [20,35,36].

Th e psychrotrophic nature and the ability of the S. putrefaciens to reduce TMAO to trim-ethylamine (TMA) explain its importance in spoilage of fi sh stored at low temperatures where the “fi shy” off -odor of spoiling fi sh is caused by the production of TMA. Th e bacterium also degrades sulfur-containing amino acids and produces volatile sulfi des including H2S [21]. H2S-producing bacteria constitute only a minor fraction of the initial microbiota on newly caught fi sh, but Gram-negative, psychrotrophic species become dominant during iced stor-age, and H2S-producing bacteria will typically grow to levels of 107 colony forming units (CFU)/g.

Table 29.3 Production of Biogenic Amines by Muscle Microbial Biota in Muscle Foods and Broths

Biogenic Amine Bacteria

Storage Condition

FactorsT (°C) Medium/Pack

PUT Hafnia alvei, Serratia liquefaciens, Shewanella putrefaciens

1 vpa pH, ornithine (arginine) utilization1 Fish broth

CAD H. alvei, S. liquefaciens, S. putrefaciens

1 vp pH, lysine utilization

1

HI Proteus morganii, Kebsiella. Pneumoniae, H. alvei, Aeromonas hydrophila, Moraxella morganii, Photobacterim phosphoreum, S. putrefaciens

1.7 Fish in vp Temperature, pH, histidine utilization

2.1 Fish in vp, fi sh broth

SPM pH, SPD

SPD pH, agmatine, arginine

TY Lactobacillus sp., L. carnis, L. divergens, Enterococcus feacalis

1 vp pH

20 airb

Tryptamine pH

Sources: Bover-Cid, S. et al., Eur. Food Res. Technol., 216, 477, 2003; Chaouqy, N.E. et al., Sci. Aliment, 25(2), 129, 2005; Dainty, R.H. et al., J. Appl. Bacteriol., 63, 427, 1987; Emborg, J. et al., Int. J. Food Microbiol., 101, 263, 2005; Karpas, Z. et al., Anal. Chim. Acta, 463, 155, 2002; Lopez-Caballero, M.E. et al., Eur. Food Res. Technol., 215, 390, 2002; Luten, J.B. et al., Quality Assurance in the Fish Industry, Huss, H.H. et al. (Eds.), Elsevier, Amsterdam, the Netherlands, 1992, 427–439; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140; Veciana-Nogués, M.T. et al., J. Agric. Food Chem., 45, 2036, 1997.

a Vacuum pack.b Aerobic storage.



Table 29.4 Genera of Microorganisms Commonly Found on Seafood

Microorganisms Gram Reaction Fish

Bacteria

Acinetobacter − X

Aeromonas − X

Alcaligenes − X

Alteromonas − X

Bacillus + X

Brochothrix + X

Chromobacterium − X

Corynebactenum + X

Cytophaga X

Enterobacter − X

Enterococcus + X

Flavobacterium − X

Halobacterium − X

Lactobacillus + X

Microbacterium + X

Moraxella − X

Morganella − X

Photobacterium − X

Pseudomonas − XX

Shewanella − X

Staphylococcus + X

Streptococcus + X

Vibrio − X

Weissella + X

Yersinia − X

X = known to occur; XX = most frequently isolated.



Th e appearance and physical properties of the fi sh body change due to action of the microorganisms. Besides odor and fl avor the slime on skin and gills, initially watery and clear, becomes cloudy, clotted, and discolored. Skin loses its bright irridescent appearance, bloom and smooth feel becomes dull, bleached and rough to the touch [4,18].

29.2.3 Physicochemical Spoilage—Fat OxidationOxygen promotes several types of deteriorative reactions in foods including fat oxidation, brown-ing reactions, and pigment oxidation. Th ere is a wide surface area exposed to oxygen on fresh fi sh. Fish lipids are rich in the polyunsaturated fatty acids called omega-3 fatty acids. Th e lipids in oily fi sh are concentrated in a layer beneath the skin in contiguity with the dark muscle bands which contain powerful oxidizing enzymes such as cytochrome c. Oxygen reacts with this fat to produce rancidity, and undesirable strong odoriferous and fl avorous compounds are produced. Fish spoil rapidly in air due to moisture loss or uptake, reaction with oxygen, and the growth of aerobic micro-organisms i.e., bacteria and moulds. Fat oxidation can be a serious problem for frozen fi sh stored for very long. Th is is one reason why fatty fi sh like bluefi sh do not remain in good condition during frozen storage unlike leaner fi sh like fl ounder [27].

29.2.4 Enzymatic SpoilageDigestive enzymes may begin to digest the fi sh itself, causing belly burn or softening of the fl esh around the gut. While the quantity of the enzymes rises, autolytic changes occur and fl avor com-ponents make the fl esh tasteless. Th is is especially likely if a fi sh is caught while feeding, since its digestive enzymes are already active. Other enzymes in fi sh muscle can also begin to aff ect the fl avor and texture of the fi llet. At warmer temperatures, the enzymes act more rapidly. But even at low temperatures, enzymatic processes may lead to a product showing a high degree of proteolysis. As a consequence of enzymatic decarboxylation of amino acids due to microbial enzymes and—to a lesser extent—to tissue activity, biogenic amines accumulate in fi sh, and other foods. Main biogenic amines usually found in fresh and processed meat products are putrescine (PUT), cadav-erine (CAD), histamine (HI), and tyramine (TY) while natural polyamines (PAs) spermidine (SPD), and spermine (SPM) content slightly changes during storage or processing [24].

29.3 Determination MethodsA well-established method for the evaluation of fi sh freshness is the sensory method while quality index method (QIM) is also used as a reference method [52]. QIM is based on well-defi ned char-acteristic changes of quality-related attributes (eyes, skin, gills, and smell) and corresponding score system of index points. Th e activity of micro-organisms is the main factor limiting the shelf life of fresh fi sh resulting in the degradation of the fi sh and the development of off -fl avors. Th e estimation of the total viable counts and measurement of chemical indicators are used as acceptability indices in guidelines and specifi cations. Sensory and microbial analyses are lengthy procedures, so rapid chemi-cal, biochemical, and physical methods are better solutions to measure the freshness of fi sh. Th ese methods are based on e.g., nucleotide catabolism or production of amines, or physical properties e.g., electrical properties by handheld devices. However, none of these methods are widely used in fi sh industry [51]. In recent years, research has focused on developing new, rapid instrumental methods



to detect the freshness of fi sh. Several new promising techniques have been developed and some of them have shown good correlation with traditional methods evaluating quality of freshness [22].

29.3.1 Sensory AnalysisSensorial analysis employs several criteria, including the appearance of the skin, eyes, mucus and gills, the fi rmness of the fl esh, and odor. Th ese criteria are checked by trained specialists with evaluation equipment. Th ey determine the samples using appropriate common, universal food sensory analysis. Th ese are wholly dependent upon the human senses: sight, touch, odor, and fl avor. Measure of a particular aspect of quality is usually compared to a standard, having gradations of quality (a well-known scale). It is always possible to construct a scale showing change or incidence by the use of value words: slight, trace, medium, moderate, very highly. A further development of scales is to denote the diff erent steps by numbered scores from 0 or 1 upwards. Human senses are better at recognizing complexities and are more discriminatory than instruments, but such determination of freshness is in part subjective, because of personal expressions. In addition, for transformed products, such as fi llets, sensory analysis is less reliable because the number of assessment criteria diminishes [4,5].

29.3.2 Total Volatile Bases (TVB) and Trimethylamine (TMA) Determination

Th e procedure of this method is to extract nitrogen, the increase of which is one of the causes of fi sh spoilage, and then express the result as mg of nitrogen in 100 g of sample. During the extraction the sample must be homogenized fi rst, then trichloroacetic acid (TCA) is added, and fi ltered. Th e extract is placed in a Conway plate and total volatile bases (TVB) are determined according to Conway’s microdiff usion method. Th e method is based on an osmotic mechanism and capillary electrophoresis. Volatile basic nitrogen (VBN) is collected with hydrochloric acid solution.

Flow injection analysis (FIA), gas diff usion cell, and a laboratory-built photometer monitor TMA and total volatile bases (TVB) in fi sh sauce. Peak of TMA is infl uenced by sample matrix.

29.3.2.1 Solid-Phase Microextraction

Solid-phase microextraction (SPME) is a proven tool in volatile analysis and has previously been used for the analysis of fl avor volatiles in seafood as well as in a variety of other foods. In general, SPME is both simple and cost-eff ective to use and can be used to analyze the levels of a wide range of volatile compounds.

Th e method was developed for the analysis of salmon volatiles using SPME and gas chroma-tography (GC)–mass spectrometry (MS).

Th e levels of several of the volatile compounds change during storage. Th ere are several alcohols and aldehydes identifi ed as potential markers for salmon freshness (e.g., cyclopentanol, hexanal). Some other volatiles (acetoin, ethyl benzene, propyl benzene, styrene, 3-methyl butanoic acid, and acetic acid) were identifi ed as potential markers for salmon spoilage. Th e method can be used manually with any GC or GC/MS. GC can separate volatile and semivolatile compounds with great resolution, but it cannot identify them. MS can provide detailed structural information on most compounds and identify them, but it cannot readily separate them. Th is contributed to an idea of the combination of the two techniques. In both techniques, the sample is in the vapor phase, and both techniques deal with about the same amount of sample (usually less than 1 ng). Th e vapor phased sample is carried



by the gas that is almost always a small molecule such as helium or hydrogen with a high diff usion coeffi cient. In MS method, organic molecules have much lower diff usion coeffi cients.

GC effl uent (the carrier gas with the organic analytes) is sprayed through a small nozzle because of the high diff usion coeffi cient; the helium is sprayed over a wide solid angle. And in MS spectrom-etry the heavier organic molecules are sprayed over a much narrower angle and tend to go straight across the vacuum region. Th en the gas passes it to packed columns of the mass spectrometer. Th e higher-molecular-weight organic compounds are separated from the carrier gas, which is removed by the vacuum pump.

Th e compounds of the sample are separated by the special separating material inside (e.g., separators made from glass drawing down a glass capillary). Th e particles escape from the column and are lodged in the spray orifi ce and stop (or at least severely reduce) the gas fl ow out of the GC column and into the mass spectrometer.

All modern GC–MS data systems are capable of displaying the mass spectrum on the computer screen as a bar plot of normalized ion abundance versus mass-to-change (m/z) ratio (often called mass). Like the other parts of the GC–MS instrument, the data system must be calibrated [31].

FIA appears to be an adequate methodology for quality control in fi sh sauce production with the advantages of reduced sample volume, energy consumption, and cost. Unfortunately, there is a major incompatibility between the two techniques: Th e compound exiting the gas chromatograph is a trace component in the GC’s carrier gas at a pressure of about 760 torr, but the mass spectrom-eter operates at a vacuum of about 10−6 to l0−5 torr. Th is is a diff erence in the pressure of eight to nine orders of magnitude, a considerable problem.

29.3.2.2 HS/MS Analysis

Samples are equilibrated at room temperature by immersion in a water-bath for 30 min and homogenized for 1 min. Th e sample is washed and stored at −80°C before the analysis. To elimi-nate variations due to time spent in the automatic sample carousel, each vial is stored at 4°C and then equilibrated for 10 min at room temperature before analysis. To minimize the infl uence of the column of a gas chromatograph, the oven temperature is set at 220°C to permit simultaneous elution of compounds and direct observation of the abundance of ions.

29.3.3 Detection of Fish Spoilage by Colorimetry—Texture Assessment

A colorimetry is a rapid method for evaluating the degree of bacterial degradation of fi nfi sh (such as codfi sh, catfi sh, and winter fl ounder). It can be carried out quickly by any lay person, with minimum training, and with portable supplies. It can be also carried out in any environment, e.g., on a fi shing boat, without interference from the odor of the environment.

What is very important—test results are objective and visually demonstrated by a color comparison which is clear to any lay person without the need for special training or evaluation equipment. A simplifi ed colorimetric test process is based upon the discovery that triphenyl tetra-zolium dye salts are colorless, ionized, water soluble, and capable of passing through the cell wall into a bacterial cell while undergoing a reduction reaction to form a nonionic, water-insoluble, and red-colored triphenyl tetrazolium formazan compound which is deposited within the bacterial cells. Th e intensity of the formed color is proportional to the concentration of the bacteria present, necessary to produce the reduction reaction, and therefore the visible color intensity provides a



measure of the bacterial concentration which, in turn, provides an indication of the quality of the fi sh being tested.

In the case of dark-fl esh fi nfi sh, such as tuna fi sh, additional reactants such as an oxidizing or bleaching agent, preferably hydrogen peroxide and a defoamer are added to bleach the muscle pig-ments and lighten the color of the solution so that any color that appears during the assay is due to the reduction of the tetrazolium dye.

Th e intensity of the color of the solution is compared by a visual colorimetric comparison with control color strips representative of known concentrations of formazan solution ranging from sub-stantially colorless, for a product of excellent quality, light reddish color representing good quality, darker red color representing borderline quality, and intense red color representing unacceptable quality. Th ese control colors are preliminarily determined by known traditional methods used to measure the bacterial content of fi nfi sh of excellent, good, borderline, and unacceptable freshness, such as total plate count determination, TMA determination, and sensory (odor) determination. Most of these traditional methods are not practical and need special, demanding environment.

Ammonia is one of the compounds produced as fi sh or fi sh-products spoil. Th e study of the sensor response to standard ammonia gas provided a model of the response to total volatile base nitrogen (TVB-N). If amines and ammonia are produced during fi sh spoilage, the concentration TVB-N in the headspace is likely to be suffi ciently high so that it can be detected by this proposed sensor (colorimetric sensor). In this method synthetic ammonia gas in nitrogen is used in the colo-rimetry to characterize the sensor. Further dilution of the ammonia gas with nitrogen is achieved through the use of mass fl ow controllers. Th e sensor is placed into a fl ow cell fi tted with the optical scanner, which monitors in real time the sensor responses to changing ammonia concentration. A PC connected to the optical scanner log the data [53,54].

29.3.4 Quantifying Biogenic AminesTh e determination of biogenic amines in fresh and processed food is gaining great interest not only for their potential risk for human health, but also because they could have a role as chemi-cal indicators of unwanted microbial contamination and processing conditions. Biogenic amines are indicators of fi sh spoilage because their precursor amino acids are decarboxylated by bacterial enzymes (Table 29.3). We can monitor the presence of diacetyl by exposing an aromatic ortho-diamine at acidic pH to an environment containing, or possibly containing, diacetyl and detecting any change in the absorption or refl ection of electromagnetic radiation due to the ortho-diamine. Th e change may suitably, though not necessarily, be in the UV or visible region or both. At fi rst a previous confi rmation is necessary, and then an extraction with hydrochloric acid [5].

With high-performance liquid chromatography (HPLC), analytes are carried through a column fi lled with packing material (stationary phase) via a mobile phase (e.g., organic solvent). Individual analytes have varying affi nities for the stationary phase and have characteristic retention times that dictate when each component will elute. Components coming off the column are visualized with a detector, e.g., fl uorescence. Th is monitors eluents based on the ability of the sample compound to absorb light at a specifi c wavelength. Unfortunately, there are drawbacks of these methods that are related to precolumn, postcolumn, or on-column derivative process leading to an overall long analysis time, and low reproducibility owing to the stability of the derivatization products. Pre derivatization consists of a series of manual time consuming steps that may introduce imprecision but off ers certain selectivity. Postcolumn derivatization has the advantage that it is in line, but it adds complexity to the instrumentation, and system must be set up in order to reduce the contributions to band widening. Changing of pH with a postcolumn system is simple, easy, and quick.



Th e example of the chromatographic technique is ion-pair chromatographic method on a C18 reversed-phase column involving a postcolumn reaction with o-phthaldialdehyde (OPA) to form fl uorescent derivatives with amines. For HI, European Community and Spanish regulations have fi xed a maximum average value of 100 mg/kg in a group of nine samples of fresh or canned fi sh and lower than twice this value for ripened fi sh products. Th e U.S. Food and Drug Adminis-tration have lowered the HI defect action level from 100 to 50 mg/kg.

29.3.5 Microbiological AnalysesBacterial growth is a determinant factor in shell fi sh. Th e microbiota that is responsible for fi sh spoilage is formed by Gram-negative bacteria. Microbial spoilage is identifi ed by the appearance of colonies. Certain spoilage metabolites can be used as quality indices. Th e shelf life of fi sh and shellfi sh is correlated to the level of specifi c spoilage bacteria and not to the count of total viable organisms, as it was thought before. Spoilage is organoleptically detectable when the number of sulfi de producing bacteria exceeds 107 CFU/g of fi sh muscle (Table 29.5).

Th e bacteriological production of hydrogen sulfi de is a very common cause of spoilage in a variety of foods such as chilled fi sh.

A number of attempts have therefore been made to detect the H2S-producing bacteria and various media have been developed. Th e example is a peptone iron agar (PIA) based on the detec-tion of H2S-producing bacteria which appear as black/gray colonies due to the precipitation of sulfi de complexes which are formed when H2S produced from thiosulfate reacts with metal ions such as Fe2+ or Pb2+. Th e number of black colonies on cysteine containing IA is, therefore, a good indication of the number of spoilage organisms in wet fi sh.

Table 29.5 Specifi c Spoilage Microbiota Dominating on Fresh Fish Stored at 0–4°C under Different Gas Atmospheres

Gas Composition Fish

Air S. putrefaciens, Pseudomonas spp.

>50% CO2 with O2 B. thermosphacta, S. putrefaciens

50% CO2 P. phosphoreum, lactic acid bacteria

<50% CO2 with O2 P. phosphoreum, lactic acid bacteria,

100% CO2 B. thermosphacta lactic acid bacteria

Vacuum packaged Pseudomonas spp.

Sources: Based on Gram, L. and Huss, H.H., Int. J. Food Microbiol., 33, 121, 1996; Huss, H.H., Assurance of Seafood Quality, FAO Fisheries Technical Paper, No. 334, United Nations Food and Agriculture Organization, Rome, Italy, 1995a; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140.



29.3.6 Rapid Methods for Detection and Quantifi cation of Microorganisms

Detecting and enumerating sulfi de-producing bacteria (SPB) is based on bacterial growth. SPB, such as S. putrefaciens, are present in seawater and on the surface of all living fi sh and shellfi sh, and are transferred to the fl esh during catch and processing. Th us they are especially responsible for fi sh and fi sh products spoilage, even when the fi sh are stored on ice.

A growth medium containing iron and sulfur is combined with the food sample forming an incubation mixture which is incubated for a period of time. SPB are determined in the sam-ple if the fl uorescence measurement initially increases and then decreases to form a fl uorescence maximum (peak). Th e time to detect the fl uorescence peak can be used with a correlation sched-ule to enumerate the SPB in the food sample. A visual test can also be used to identify color changes in the incubation mixture to provide a semiquantitative enumeration of SPB eff ective after incubation.

29.3.6.1 Short-Wavelength Near-Infrared Spectroscopic Method (SW-NIR)

In this method, the key to determine fi sh spoilage and to quantify microbial loads is to use visible and short-wavelength near-infrared (SW-NIR: 600–1100 nm) spectroscopy. In the SW-NIR region, various fundamental molecular vibrations, including those from CH, O–H, N–H, C=O, and other functional groups can be detected [42]. If a fi sh sample is irradiated with NIR light, it absorbs the light with frequencies matching characteristic vibrations of particular functional groups, whereas the light with other frequencies will be transmitted or refl ected. Th erefore, the biochemical components of a food tissue determine the amount and frequency of absorbed light and the quantity of refl ected or transmitted light can be used to infer the chemical composition of that food tissue [42].

29.3.7 Predicting Fish SpoilageTh e basic question to be addressed among scientists dealing with predicting modeling is “how wrong do they have to be not to be useful?” Mathematical models have been developed for the quantifi ca-tion of parameters, e.g., temperature, aw, pH, and carbon dioxide that aff ect growth and survival of spoilage bacteria such as B. thermosphacta, lactic acid bacteria, P. phosphoreum, pseudononads, S. putrefaciens, Listeria monocytogenes, and Salmonella in muscle foods [2,8,10,12,13,39,46,47,57,59]. However, the application of these models has not been focused on monitoring muscle food quality per se, but mainly as a management tool for shelf life and safety prediction and as a scientifi c tool to gain insight into muscle food spoilage.

Diff erent models (kinetic or stochastic) have been developed for various muscle products but still an accurate prediction of shelf life is not attainable [15,33,34,56,59,61,62]. Th ere are limited, successfully validated models for predicting the growth of ESOs that have been included in appli-cation software and this has facilitated the prediction of food shelf life under constant and dynamic temperature storage conditions (Table 29.6). Measurement of enzyme synthesis and activity could be used to estimate shelf life, off ering a completely diff erent modeling approach [58].

Th e quality of the fi sh depends on how far the normal spoilage processes have progressed. Th ese processes cannot be stopped, but the rate at which they occur can be controlled. Th e most



important thing is to control the storage temperature—fresh, unfrozen fi sh should be kept as close as possible to 0oC, so the best way to do that is to pack fi sh in ice or ice water. Th e predic-tion can mean acidifi cation or the addition of preservatives like sorbate and benzoate. Also drying or heavy dry-salting of fi sh eliminates bacterial growth. Recently preservation of fi sh shelf life has relied on the elimination or growth inhibition of spoilage organisms because the growth of bacteria is exponential. Respiratory Gram-negative bacteria are typically inhibited in fi sh products preserved by the addition of low levels of NaCl, a slight acidifi cation and chill storage in vac-uum packs. Under these conditions, the microfl ora typically becomes dominated by Lactobacillus spp. and Carnobacterium spp. with an association of Gram-negative fermentative bacteria such as P. phosphoreum and psychrotrophic Enterobacteriaceae.

Growth of the ESOs can help in prediction from fi sh spoilage, but the technique that iden-tifi es the bacteria must be capable of detecting as little as 102 ESO/g of product. Shelf live can be predicted as the time ESO require to multiply from initial number to the minimal spoilage level (MLS).

An example of the ESO’s technique is the CO2 packing of fresh fi sh. Th e respiratory spoilage bacteria (Shewanella and Pseudomonas) are inhibited then and the shelf life is markedly extended [14,18].

Table 29.6 Available Shareware Software for Fish Spoilage

Seafood Spoilage and Safety Predictor (Shelf life of seafoods and growth of ESOs; Listeria monocytogenes in cold-smoked salmon) Dalgaard et al. [7].

Safety Monitoring and Assurance System (Koutsoumanis et al. [34,38]) (Greek predictive microbiology application software under development); software is based on kinetic data of spoilage bacteria derived from fi sh, meat and milk in situ.

Gri-Gri*; shelf life modeling of fi sh stored under various storage conditions (*Greek for the fi shermen setting sail in the evening) Software produced by K. Koutsoumanis ([email protected]) within G Nychas’s group ([email protected]) of the Agricultural University of Athens, Greece.

Other available software not specifi cally focused on fi sh

GrowthPredictor (UK)—www.ifr.ac.uk/Safety/GrowthPredictor/(based on data previously used in the FoodMicromodel software; 18 models for growth of pathogenic bacteria; available free of charge).

Sym’Previus—www.symprevius.net (French predictive microbiology application software under development).

Pathogen Modelling Program (USA)—www.arserrc.gov/mfs/pathogen.htm (37 models of growth, survival and inactivation; frequently updated (version 7.0); Available free of charge during the last 15 years; ~5000 downloads per year.

Sources: Based on Dalgaard, P. et al., Int. J. Food Microbiol., 73, 343, 2002; Koutsoumanis, K. et al., Int. J. Food Microbiol., 73, 375, 2002; Palleroni, N.J., The Prokaryotes, Balows, A. et al. (Eds.), Springer-Verlag, New York, 1992, 3071–3085. www.arserrc.gov/mfs/pathogen.htm; www.ifr.ac.uk/Safety/GrowthPredictor/.



29.4 ConclusionsFresh fi sh is a product with a very short commercial life and high variability. After caching, unpreserved fi sh is spoiled by Gram-negative, fermentative bacteria (such as Vibrionaceae). Generally, the rates at which autolytic and microbial spoilage occur are dependent on the degree of microbial contamination and biota, storage temperature, and packaging. Fish spoils through the combined eff ects of chemical reactions, via the continuing activities of endogenous enzymes and lipid oxidation. Th e factors that infl uence the microbial contamination and growth include fi sh spe-cies and size, method of catch, on-board handling, fi shing vessel sanitation, processing, and storage condition.

Th e major cause of food spoilage is microbial growth and metabolism resulting in the formation of amines, sulfi des, alcohols, aldehydes, ketones, and organic acids with unpleasant and unacceptable off -fl avors [18].

To determine the level of fi sh spoilage, sensory and microbiological analyses are most widely used. Sensory analysis is appropriate for product development, but is costly and therefore not an attractive proposition for routine analyses. Microbiological methods give retrospective informa-tion which is satisfactory for product safety validation but unsuitable for product monitoring. Even when the ESOs are well established, there are instances where their enumeration is of limited value.

Combining microbial ecology, molecular techniques, analytical chemistry, sensory analysis, and mathematical modeling allows us to characterize the ESOs and to develop methods to deter-mine, predict, and extend the shelf life of products. Enumeration of ESO bacteria requires the development of rapid methods because 2–4 days are not reliable to determine the quality of a very perishable product such as fi sh. More rapid alternatives are being developed, e.g., epifl uorescent microscopy, fl ow cytometry, and electrical impedance.

Compared with microbiological methods, which are slow, chemical analyses may be signifi -cantly faster; however, for some compounds measurable concentrations are not present until close to spoilage. Smart packaging and use of fi eld-validated indicators off er a new approach in detecting spoilage by the consumer [15,16].

Th e spoilage can be predicted in fi sh and fi sh products preserved by the most commonly used methods like low temperature storage, dehydration, canning, MAP, irradiation, the use of chemi-cal and biological preservatives, and combinations of two or more of these methods. All these contribute to the improvement of quality and safety of seafood.

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