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ROGÉRIO MENDES

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GUIDEBOOK ON MELANOSIS INHIBITORS AND PROCESSING TECHNOLOGY OF CRUSTACEANS

Introduction ................................................................................................................................ 3 Crustacean species...................................................................................................................... 3 Melanosis inhibitors ................................................................................................................. 13

Sulphites ............................................................................................................................... 13 4-Hexylresorcinol................................................................................................................. 14

Handling shrimp onboard......................................................................................................... 15 Recommendations ................................................................................................................ 18 Careful and Hygienic Handling............................................................................................ 18

Methods of inhibitors application ............................................................................................ 18 Dusting ................................................................................................................................. 19 Dipping................................................................................................................................. 20 Ice made with inhibitors solutions ....................................................................................... 20 Inhibitors in refrigerated sea water....................................................................................... 20

New Processing Technologies ................................................................................................. 22 Carbon dioxide application .................................................................................................. 22

Carbon Dioxide Modified Refrigerated Seawater............................................................ 22 Pressurized Dissolved Carbon Dioxide............................................................................ 22 Soluble Gas Stabilisation ................................................................................................. 24

Liquid Ice ............................................................................................................................. 25 Regulations relative to the use of additives.............................................................................. 27

Sulphite................................................................................................................................. 27 4-Hexylresorcinol................................................................................................................. 27

Specifications for chilled and frozen crustacean...................................................................... 29 Quality control.......................................................................................................................... 30

QIM ...................................................................................................................................... 30 Analysis of Residues ............................................................................................................ 33 Sulphite................................................................................................................................. 33 4-Hexylresorcinol................................................................................................................. 33

Annex 1: Monier-Williams Method......................................................................................... 34 Annex 2: Enzymatic Method.................................................................................................... 36 Annex 3: Alert for Sulfites ....................................................................................................... 37 Annex 4: 4-Hexylresorcinol ..................................................................................................... 38 Annex 5: Quality parameters.................................................................................................... 40

Indole method....................................................................................................................... 40 ROGÉRIO MENDES rogerio@ipimar.pt

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Introduction The application of melanosis inhibitors on crustacean goes back for more than 50 years. Use of inhibitors based on sulphite is the common rule and application is frequently in a less correct way. On account of this products often present exceedingly levels of sulphites which may be dangerous for consumers, namely asthmatic ones. This guidebook contains technical advice on the handling and processing of shrimp and Norway lobster during operations onboard and on shore. Advisory information is given on what concerns the application of old and new chemicals for melanosis inhibitory purposes, chilling, freezing, cold storage, processing techniques and safety concerns affecting the quality of the products. It is not expected that this guidebook will answer all the questions about inhibitors and processing technology of crustacean, nevertheless it will provide a contribute for the safe use of food additives and production of better quality products. If the enclosed procedures are followed healthier products with increased shelf life will be available for consumers.

Crustacean species In general, there is a common recognition of the bad reputation of crustacean from the south of Europe to develop melanosis in comparison with crustacean from other European fishing ground, namely from the north. This is probably mainly because of the higher temperatures on deck after landing and during sorting and processing. These crustaceans are therefore the most prone to bad handling and for this reason the majority of the knowledge and recommendations presented are particularly directed to their processing. Crustaceans in the south of Europe include mainly three species, deepwater pink shrimp (Parapenaeus longirostris), red shrimp (Aristeus antenatus) and Norway lobster (Nephrops norvegicus). For a better knowledge of these species elements concerning their composition and nutritive value are present in Figure 1, being the data from Bandarra et al. (2004).

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Figure 1 – Composition and nutritional value of major crustacean species caught in the South ofEurope. In: Bandarra, N., Calhau, M.A., Oliveira, L., Ramos, M., Dias, M.G., Bártolo, H.,Faria, M.R., Fonseca, M.C., Gonçalves, J., Batista, I., Nunes, M.L., 2004. Composição evalor nutricional dos produtos da pesca mais consumidos em Portugal. PublicaçõesAvulsas do IPIMAR, 11, 100 p.

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Figure 2 – Continuation of Fig 1.

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Figure 3 – Continuation of Fig 1.

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Figure 4 – Continuation of Fig 1.

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Figure 5 – Continuation of Fig 1.

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Figure 6 – Continuation of Fig 1.

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Melanosis or black spot, what is it?

Browning is one of the major problems in the food industry and can cause deleterious changes in the organoleptic properties of foods, resulting in shorter shelf life and quality, and therefore a decrease in commercial value. It is the case of crustaceans. Melanosis, or black spot, in crustacean is a condition in which the membrane between the shell and the tail muscle darkens as a crustacean deteriorates. Melanosis or black spot development is a cosmetic problem and not a health hazard. Black spot typically begins forming on a fresh shrimp at the base of the legs, spreads over the carapace and proceeds down into the tail region. Melanosis starts in refrigerated and frozen crustaceans within just a few hours of capture, and susceptibility is often greater in autumn and winter, coinciding with moulting and lower food availability. The reaction was once thought to be microbial in nature but has since been demonstrated to be an enzymatic process. Refrigeration or storage on ice slows down but does not prevent this reaction because the enzymatic process remains active even under these conditions.

Origin of melanosis

During the development of a new shell in a moulting crustacean, tyrosine is an amino acid that is part of the material needed for the production of the new shell. This molecule is therefore the base for the development of melanosis. During the intermoult period, crustaceans accumulate tyrosine under the shell in the tail and head. Melanosis in crustaceans is a natural postmortem mechanism that involves the action of an enzymatic complex, polyphenoloxidase (PPO) a copper-containing oxidase, which in the presence of oxygen acts on naturally-occurring colorless phenols, thus forming compounds which can polymerize into insoluble dark melanine pigments. Subsequently a non-enzymatic polymerization of the quinones occurs, leading to the formation of dark, high molecular weight pigments. This same series of reactions is responsible for enzymatic browning in fruits and vegetables. PPO is unique in that it catalyses two different types of reactions involving phenolic compounds. The first reaction involves hydroxylation of monophenols (such as tyrosine) to produce o-diphenols, and in the second reaction PPO oxidizes o-diphenols to o-quinones (Fig 7). After the death of crustacean PPO becomes active and oxidizes in a first moment tyrosine in DOPA (dihydroxyphenylalanine) or in derived DOPA products (dopaquinone). Two other enzyme (dopadecarboxylase) and transacetylase) synthesize also DOPA derived products (N-acetyldopamine). The derived products formed are very i«unstable molecules and are easily oxidized. This oxidation is catalyzed by light. Through molecular rearrangement (polymerization) a coloured compound is formed, the melanine. The PPO, necessary for the hardening of the shell, is present in the shell, antenna and blood. When the crustacean dies, this enzyme is still active and converts tyrosine to melanin, binding onto protein. Once the pigment is bound, it cannot be removed without destruction of the protein it is attached to. While the crustaceans are alive this mechanism is under control and melanosis will not

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develop, except where there is an injury. Black spots (melanosis) form on crustacean shell within a few hours after capture and also after thawing.

Figure 7 – Mechanism of development of melanosis in crustaceans. The pigments themselves are not dangerous to human health, but even so affected crustaceans are rejected by consumers because of their appearance (Fig 8). Sulphite-based formulations, mainly metabisulphite, are currently used to avoid or at least delay melanosis. High concentrations of additive are sometimes required to prevent melanosis effectively. For this reason it is relatively common for local seafood retailers to add an extra dose of metabisulphite, thereby increasing the total content in the edible portions to levels exceeding the limits established by lawmakers and regulators.

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Figure 8 – Deepwater pink shrimp (Parapenaeus longirostris) with (right) and without (left) melanosis.

Controlling factors

As previously referred the melanine is not produced by any microbiological phenomena. The most significant factors involved in the development rate of melanosis are:

- an increase of the pH of the muscle tissue. The PPO is active in basic medium and inactive in acid medium,

- increased temperature. Cooking reduce significantly the enzyme activity and well as freeze storage,

- the level of substrate. The higher the free tyrosine existent in the body of the organisms the higher the melanosis development,

- the level of enzyme. Particularly PPO level in the blood, carapace, head and legs,

- presence of oxygen. Increased development of oxidation reactions, - presence of copper, co-factor for the enzymatic reaction of PPO - copper binding agents such as EDTA (ethylenediaminetetraacetic acid-

E385) inactivate PPO. Inversely addition of copper enhances PPO activity. - species of crustacean plays an important role in the quantity of enzyme

and substrate, - sex, - season, particularly the period of molt, - geographical origin of crustaceans, - technological factors. Adequate care at the moment of catch, namely

temperature and exposure to light

The control of these factors by rapid handling on deck can reduce significantly the level of incidence of melanosis. That said, Good Manufacturing Practice aboard vessels has been shown to play an important part in delaying the onset of black spot formation in prawns. Furthermore the application of chemicals which interfere with the enzyme reaction will prevent melanosis.

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Melanosis inhibitors

Sulphites

Sulphite chemicals are food additives belonging to the class of preserving agents (E220 to E228). These molecules have been used in the last 50 years to prevent enzymatic darkening of food products. Traditional sulphites treat the symptoms of melanosis formation. Sodium metabisulphite (Na2S2O5 – E223), commonly known as meta, is the most used sulphite in the control of crustaceans melanosis. The sulphites added to food products by competing with tyrosine act as inhibitors of PPO preventing the development of colored components (quinones) and thus the formation of melanine. This type of chemicals in the presence of oxygen act by releasing sulphur dioxide (SO2) which forms sulphite when dissolved in water. It is the sulphite that lowers the medium pH and bonds to compounds that produce melanine, thus inhibiting the enzyme reaction and preventing the development of the pigment.

Sodium metabisulphite is available as a powder in various commercial forms (Figure 9) and the recommended use is to dissolve it in water while dipping the crustacean in it for a certain period of time while at sea. As sulphites become consumed, additional sulphite treatment is required to prevent the melanosis forming. Furthermore, sulphites lose their effectiveness over time as residual sulphite is diluted by melting ice and washes off. It is important to note that shrimp frozen onboard is normally highly susceptible to melanosis after thawing.

This is easily explained in that the shrimp even when treated with sulphite are immediately frozen after catch. The compounds and enzyme responsible for melanosis are still in the shrimp, however, they are not highly active under frozen conditions. As the shrimp thaw, residual sulphite that may be present can be diluted by the melting water released and rinsing of the shrimp. Since some of the highly reactive compounds were already present prior to freezing the melanosis proceeds at what seems to be an accelerated rate.

It is well known that sulphites produce certain adverse reactions in some groups of population, mainly asthmatic. The FDA estimates that one of a hundred people

Figure 9 – Commercial form of sulphites used for prevention of melanosis in crustacean availablefrom TURCO, Spain.

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is sulfite- sensitive, and that 5 percent of those with asthma are also at risk of suffering an adverse reaction to sulfites. A person can develop sulfite sensitivity at any point in life, and no one knows what triggers onset or the mechanism by which reactions occur. In fact, metabisulphite is considered as a precipitating cause of an asthmatic attack. Because of this, their used in food is limited, making research into alternatives to sulphite derivatives very important. There is therefore a need to find alternatives that will inhibit melanosis formation, avoid health safety issues for consumers, and retard microbial growth. To date, 4-hexylresorcinol, benzoic acid, kojic acid, cumic acid, or ethylenediaminetetraacetic acid (EDTA) have proved to be partially effective melanosis inhibitors. When used in the right concentrations, these chemical agents are capable of stopping melanosis formation nearly completely. 4-hexylresorcinol appears especially to be a good alternative. Its use is permitted in the US, Canada, Australia, and some Latin American countries. Nevertheless, the proportions required for inhibiting melanosis have not been clearly established. Some workers have cited 50 ppm, other researchers have reported 100 ppm, while still others have cited considerably higher concentrations, namely, as much as 0.5 % and 0.1 % in deepwater pink shrimp. These differences may be due to interspecies variations, to cyclical changes in physiological susceptibility, or to melanosis inhibitor concentration and the method of application employed. However, nearly all reports have involved small-scale experimental or laboratory studies. The literature contains no studies on different methods of application, seasonal variations, or other factors that might influence melanosis development. For all these reasons, seeking more potentially effective 4-hexylresorcinol-based formulations holds out considerable interest.

4-Hexylresorcinol

4-Hexylresorcinol (C12H18O2) is a dihydroxybenzene with a hexyl group in the 4 position and hydroxyl groups on positions 1 and 3 of the aromatic ring. 4-Hexylresorcinol has a long history of use in cosmetics and pharmaceuticals. 4-Hexylresorcinol is proposed for use as a processing aid for prevention of melanosis in shrimp. This inhibitor is proposed as an alternative to the use of sulphites known for their problems associated with allergic reactions. Opta (Bedford, MA, USA) produces a commercial product EVERFRESH® (Fig 10) for inhibition of melanosis development in shrimp. The most widespread current use is as a topical antiseptic in throat lozenges, soaps and hand washes, and skin/wound cleaners. 4-Hexylresorcinol was widely used for many years as an extremely effective anthelmintic, prior to recent development of more selective anthelmintics (Rossof, 1974).

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Figure 10 – 4-Hexylresorcinol in commercial form produced by Opta (Bedford, MA, USA) for inhibition of melanosis development in shrimp

4-Hexylresorcinol is a functional sulfite alternative as it is a specific inhibitor of polyphenol oxydase, as compared to sulfites which react chemically with melanosis precursors. The functionality of 4-hexylresorcinol as a shrimp melanosis inhibitor has been demonstrated both in laboratory test with previously frozen shrimp and in field trials with fresh shrimp (Otwell et al., 1991). Sulphite use essentially reverses the reaction where the PPO and oxygen (O2) form the highly reactive compound. In this application the sulphite is treating the symptom of melanosis formation since the highly reactive compounds are being formed and sulphide is simply turning them back into less reactive colorless compounds. Oppositely to sulphide, 4-hexylresorcinol has a prevention role in melanosis. 4-Hexylresorcinol is a small molecule that interacts or binds specifically to the PPO enzyme thereby rendering it incapable of catalyzing (or taking part) in the reactions. If one were to remove PPO from the melanosis reaction mechanism, none of the reactions would ever take place and thus would never be the generation of highly reactive compounds to produce brown polymers.

The binding of 4-hexylresorcinol as indicated above is quite specific to the PPO enzyme. This binding is also irreversible. Therefore, once the PPO enzyme is locked by 4-hexylresorcinol, the occurrence of melanosis will nor proceed irrespective of rinsing or thawing.

Handling shrimp onboard

The good quality of the catch starts with care before the cod ends comes on board of the trawler. The trawl should last long enough to get a reasonable catch but unnecessary long tows can damage the crustacean in the net. A common practice between fishermen is to give the crustacean a preliminary wash by towing the trawl near the surface just before hauling.

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After opening of the cod end the crustacean must be handled quickly and carefully. Exposure to sun and wind on the deck must be avoided, in order to prevent spoilage which otherwise will be increased and chilling rendered more difficult. Processing of catches should be done preferably in the lower deck away from direct sun light and wind action. If impossible to comply with this installation of a canopy during warm weather is recommended.

1. After landing on deck, catch is first sorted by hand and fish, damaged crustacean and rubbish are removed (Fig 11).

2. Wash the crustacean thoroughly using a good flow of water and agitating them in the basket (Fig 12). This operation removes mud and organic matter which binds to the melanosis inhibitors and reduces the anti melanosis treatment efficiency. The washing step further removes bacteria which could contaminate other batches during dipping for inhibitors application and during chilled storage.

Figure 11 – Sorting of trawl catch after landing on board.

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3. Apply the melanosis inhibitors by dipping into the appropriate solution. Use a treatment tank and baskets made of plastic as some inhibitors react with metals. Dissolve the inhibitors according with instructions of manufacturer. Usually the solution is prepared with seawater as most of the work is performed out at sea, but the use of freshwater is just as appropriate.

4. Spread the inhibitor evenly over the surface of the water to avoid clumping and mix it thoroughly. Take care that it should be completely dissolved before immersion of crustacean is carried out.

5. Place the baskets of washed crustaceans into the inhibitors solution. Agitate gently the baskets while in the solution to ensure an even exposure of the crustaceans to the chemicals. Follow strictly the times recommended by the manufacturers of the inhibitors. Use a timer/stop watch or a watch which has a second hand.

6. After immersion, allow the products to drain to remove excess of solution during about 5 min. Proceed with packaging and freezing or with chilled storage.

Figure 12 – Washing of crustaceans with seawater after sorting.

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Recommendations

Trawled Nephrops norvegicus, Parapenaeus longirostris or Aristeus antenatus, are highly perishable and need to be promptly iced at sea and landed as soon after catching to ensure high yields of best quality product.

If the treatment solution becomes cloudy with use make up a new one as its inhibitory capacity may have been reduced by insufficient washing before dipping.

Cover the treatment solution when not in use. Exposure to sun or wind may reduce the efficiency of the inhibitors.

If using metabisulphite based inhibitors do not keep a dip overnight because sulphur dioxide evaporates.

Catches should be top and bottom iced with sufficient ice to maintain chill temperatures. For a standard 70 litre box this corresponds to an amount of about 20 kg of whole crustaceans.

The box should be not more than 200 mm deep to avoid crushing the bottom crustacean. A layer of flake ice should be placed in the bottom of the box. A layer of crustacean not more than 50 mm deep should be laid on the ice and covered with more ice. Successive layers of crustacean and ice are then added until the box is full. The boxes should be well covered with ice when they are landed. The fish room temperature should be between 1-3º C so that the ice melts slowly. Melt water should be free to drain from the bottom of the box.

Careful and Hygienic Handling

Ensure all surfaces aboard the vessel that may come into contact with seafood are hygienically clean.

Ice or ice slurry using seawater must be hygienic.

Plastic liners are recommended (but use only once).

If there is any doubt about the cleanliness of a container – DON’T USE IT.

Keep seafood covered.

Do not overfill containers.

Handle seafood with both hands.

Do not hold, handle or throw large crustacean by the tail.

Do not squeeze, squash, throw or step on seafood.

Do not use staples to attach labels to containers or fish.

Methods of inhibitors application

Methods of application are a function of the type of inhibitor considered. In general application is by immersion of crustaceans on solutions but other forms

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are possible. The main procedures will be discussed focusing mainly on the utilization of sodium metabisulphite as it is the most used melanosis inhibitor.

Because some people develop allergic reactions to chemicals avoid direct contact with skin. Furthermore, the dusting or the dipping operation should not be conducted in a confined space as the fumes of sulphur dioxide can also be health hazard.

Dusting

After washing the crustaceans sprinkle the dry product onto the crustaceans. Revolve the crustaceans in the baskets in order to spread evenly the inhibitors (Fig 13). It is suggested by manufacturers that further product should be spread in the boxes over each layer of crustaceans. Is advisable that a plastic sheet should be placed between the ice and the crustaceans in order to prevent the washing effect of melting water on the products. It should be noted that in this way part of the effectiveness of ice is lost. If no plastics sheet is used spread the metabisulphite over the ice. In general an amount around 3% is suggested by the different manufacturers. Though this method is the most widespread among fishermen, is use is NOT RECOMMENDED as it results in uneven distribution of the inhibitors resulting in some crustacean with high residues and bleaching while others will have insufficient residue to prevent melanosis. This late possibility is nevertheless less frequent, as fishermen tend to apply more sulphite than less in order to prevent any signs of melanosis.

Figure 13 – Sprinkling of crustaceans with dry melanosis inhibitors.

Raw whole deepwater pink shrimp stowed in crushed ice will keep in good condition for up to 4 days, though for best results the iced shrimp should be processes on shore within 2 days of capture. The typical shrimp flavor fades way

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completely after 6 days in ice and the meats become soft, discolored and difficult to peel. After 8 days ammonium and sour fishy odors develop.

Dipping

The immersion of crustacean for a specific time period in solutions of melanosis inhibitors (dipping) of known concentration is the best method for application of these additives. In general, and in what concerns application of sulphites, it is recommended the use of a dip containing 10 g of metabisulphite per litre (10 000 ppm) for 2-3 min (Figure 14). For each litre of solution 6 to 8 kg of crustaceans can be treated. This method allows a better homogeneity of the application of additives as well as to comply with the regulations concerning the SO2 levels. Work developed by Gomez-Guillen et al. (2005) showed however that for deepwater pink shrimp (Parapenaeus longirostris) the recommended amount of sulfites was insufficient. A dip treatment of 1 h with 50 g/kg sulfite together with citric acid (20 g/kg) and chelants, EDTA (0.45 g/kg) and PPi (30 g/kg) was shown to be effective for melanosis prevention during one week.

Ice made with inhibitors solutions

Another possibility of inhibitors application is the inclusion of the additives in the ice used in the treatment of the crustacean. This method however did not showed a clear advantage over normal ice and furthermore, the levels of chemical throughout the ice were not uniform, being reported higher concentrations in the core of the ice blocks. New equipments of ice production in which those problems are solved could present a definite progress in the way melanosis inhibitors are applied.

Inhibitors in refrigerated sea water

Though not used in the south of Europe, addition of inhibitors can also be done on the refrigerated sea water tanks. This method is not the best since local high concentrations of inhibitors can occur if no recirculation waster system is used. Furthermore, metabisulphite will react with the metal of the cooling coils causing them to corrode. The recommended precise quantities of inhibitors are difficult to define since, being function of the tank size, catch size, brine temperature and storage time, change from trawler to trawler. Shrimps stored in refrigerated sea water have been reported has having generally a better appearance than iced shrimps of the same age. A certain salt uptake should however be expected, normally around 2% by weight after 2-3 days storage, nevertheless within acceptability limits.

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Figure 14 – Application of melanosis inhibitors by dipping in metabisulphite solution. In: Whitefish,

Quality Guide. Ed. Irish Sea Fisheries Board, Irish South & West Producers Organization Ltd., Department of the Marine and Natural Resources. FIFG Project under EU Pesca measure.

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New Processing Technologies

Preservation of shellfish has involved in the last years the utilization of other systems either than the traditional use of flake ice. Apart from the application of refrigerated seawater system in preservation of shellfish, the use of carbon dioxide modified seawater and liquid ice have been the main new processing technologies applied to crustaceans.

Carbon dioxide application

Utilization of carbon dioxide has been done in conjunction with different technological approaches: carbon dioxide refrigerated seawater, pressurized dissolved carbon dioxide and soluble gas stabilization.

Carbon Dioxide Modified Refrigerated Seawater

It is well known that certain enzymes in food products contribute to their spoilage during storage over long periods. The traditional method to inactivate these undesirable enzymes is to expose the food product to elevated temperatures, which will normally destroy such enzymes. The application of heat, however, has a deleterious effect on the quality, feel or "freshness" of the food product. Thus, the elevated temperatures are not specific to enzymes but also degrade other components of the food, which contribute to desirable qualities in the food product. Other alternatives are therefore used, such as refrigerated seawater modified with carbon dioxide.

The advantages and disadvantages of the refrigerated seawater system (RSW) for holding fish and shellfish are well documented and were recently discussed by Barnett et al. (1971) and by Nelson and Barnett (1971). Based on bacteriological measurement and sensory evaluation, these authors showed that rockfish, Sebastodes flavidus, can be held in the RSW system modified by the addition of carbon dioxide (MRSW) for longer periods of time than in ice.

The application of carbon dioxide is expected to result in an irreversible inactivation of deleterious enzyme(s) in shrimp without the disadvantages attendant conventional methods until now employed for this purpose. Thus, the carbon dioxide dissolves in the aqueous phase of the food product resulting in the formation of carbonic acid. Sufficient CO2 is dissolved in the aqueous part of the food to create a carbonic acid solution having a pH sufficiently low to inactivate the enzyme(s). The carbon dioxide is allowed to remain dissolved in the aqueous phase for a time sufficient to inactivate the enzymes.

Pressurized Dissolved Carbon Dioxide

The use of CO2 under pressure is another method of exposing the food product to an atmosphere having a partial pressure of C02, and for a time, sufficient to result in dissolution of sufficient CO2 in the aqueous phase of the food product to produce a carbonic acid solution having a pH sufficiently low to irreversibly inactivate the enzyme.

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During the treatment, when the carbon dioxide is injected under pressure, it dissolves in the water, so that within the container there exist, at the same time, carbonic anhydride, the antimelanosic mixtures and of course, carbonic acid

(Fig 15). These products penetrate into the bodies of the shellfish owing to the pressure condition obtaining in the device, so that the cephalothorax, the gills, and other parts of the shellfish are completely impregnated with them. It has been found that carbon dioxide is a very good antioxidant and a digester of oxidizable substances, so that at the pressure under which the process carried out, two things occur: in the first place, the carbonic anhydride eliminates the excess of oxygen that there might still be in the shellfish, and in the second place, the carbonic acid, together with the antimelanosic mixtures, remain in the shellfish in an amount corresponding to its size. When the shellfish treated in this manner, completely impregnated with the above-mentioned products, is ice stored or frozen, the carbonic acid, frozen owing to its stability at low temperatures, serves as a preserver against possible exudations caused by changes in temperature resulting from refrigerator changes during transport, etc., thus eliminating completely the danger of renewed oxygen accumulation within the shellfish. In the meanwhile, the purpose of the acid substance (citric acid, for example) present in the body of the animal, is to maintain a pH no higher than 6.5 during the period of preservation. Experiments have shown that, preferably, the pH should be kept between 5 and 6 during preservation. The amount of carbon dioxide normally used for every liter of water is of the order of

Figure 15 – Container for preventing melanosis in shellfish with application of carbondioxide and inhibitors, according to United States Patent 3,859,450.

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12 to 15 g, while up to 3 kg of shellfish can be treated with one liter of water, with no inconvenience.

While metabisulphite and other antimelanosic mixtures are used, by itself, as a preservative, its properties and the quite imperfect absorption of the same by the shellfish make it necessary to use it in very large amounts, with its somewhat toxic nature then beginning to become apparent. If the metabisulphite is used in small amounts, then its preservative effect is nil. Therefore, the inclusion of the antimelanosic mixtures in the solution may be extremely interesting since despite its being used in minute quantities, its effects, in view of its perfect assimilation by the shellfish, which is totally impregnated as a result of the pressure to which it is subjected during treatment, are quite amazing, making it possible to lengthen the keeping time of the shellfish after thawing to a degree which, up to now, was impossible to obtain through the usual methods.

Soluble Gas Stabilisation

Soluble gas stabilization (SGS) is a novel method to increase shelf-life and maintain quality and safety of foodstuffs (Siverstsvik, 1998). SGS is part of the new processing/active packaging technologies, which include a number of diverse methods such as changing the head-space gas atmosphere (e.g. oxygen scavenging, carbon dioxide generators, soluble gas stabilization), moisture control (e.g. use of osmotic active packaging and dehydrating films), monitoring quality (e.g. time temperature integrators). SGS is a novel approach to extend the shelf-life of fish (Sivertsvik, 1999). The preservative effect of CO2 on the bacterial growth is well-known from the principles of modified atmosphere packaging (MAP), and is caused by CO2 absorption from the atmosphere into the product (Sivertsvik, 1995). In SGS the CO2 is dissolved in the product (eq. 1) at low temperature (~0º C) and elevated pressures (> 2 atm) prior to packaging, instead of as in MAP where CO2 is introduced to the package atmosphere at the time of packaging.

CO2 (g) + H2O (l) ↔ HCO3- + H+ ↔ CO 32- + 2H+ (eq. 1)

The principle of SGS is to allow carbon dioxide to dissolve into the product prior to packaging at a temperature close to 0°C and at an elevated pressure. The SGS treatment may be carried out on pink shrimp in a large chilled and gas tight steel-container before ice storage. The container is filled with 100% CO2 and the gas allowed to dissolve into the shrimp surface during 60 minutes at elevated pressure (~4 bar).

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Liquid Ice

Traditional iced storage presents some undesirable attributes (e.g. injury and bruise of the flesh and leaching of flavor components and nutritional compounds), and it can be a very expensive labor because of the size of the catch as well. Liquid ice has been introduced recently as a successful method for the rapid chilling of food products and as a way to reduce temperature of products to lower values than the attained with traditional ice (Fig 16).

On basis to its physical properties, liquid ice allows a better heat exchange power, providing a minimum melting caused by ambient heat loss. Composed of millions of microscopic spherical ice crystals suspended in seawater or brine, liquid ice prevents marking or physical damage to the seafood and at the same time maintains the preserving capacity conventionally associated to the sodium chloride presence. On the other hand, the absence of air pockets delays the biochemical processes related to the presence of oxygen (Figure 17).

Depending of the saline strength of the water and on the ice concentration in the suspension, it is possible to modify the final temperature of the liquid ice and viscosities, improving the application’s versatility and therefore, its ability to chill (Figure 18). Liquid ice maintains better food products quality, mainly by delaying both enzymatic and bacterial spoilage activity. This characteristic makes this product a very interesting alternative for fish industry, particularly because of the high perishable nature of seafood products. From a practical point of view, the main advantage of liquid ice is its pumpability, which allows the utilization of several types of containers and deposits and its application on different seafood products (Figure 18).

Figure 16 – Temperature/time change of deep water pink shrimp in liquid ice and inflake ice.

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Moreover, application on crustaceans presents an additional advantage, on account of the possibility of incorporation of melanosis inhibitors within the ice liquid fraction, thus simplifying the processing onboard. Apart from this, application of liquid ice in decapod crustaceans induces an increase in the moisture content and a consequent raise in the weight of the products. At the same time generates a massive muscular contraction which facilitates peeling out, thus avoiding the characteristic lost of the first segment of the crustaceans tail during processing.

Figure 17 – Changes in brine temperature in function of sodium chloride concentrationand ice crystal percentage in the solution.

Figure 18 - Box with liquid ice applied to deepwater pink shrimp (left) and liquid icedelivered in tubs (right).

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Regulations relative to the use of additives

Sulphite

Utilization of chemical additives in the prevention of melanosis is regulated by specific European and National regulations concerning food additives. The utilization of sulphites in the treatment of crustacean is specifically regulated by the Directive 98/72/EC of the European Parliament and of the Council that amends the Directive 95/2/EC on food additives other than colours and sweeteners (Fig 19). Allowed levels on crustacean change in function of the size and if there are raw or cooked. Different types of sulphites are authorized as food additives being the sodium metabisulphite (E223) the most used. In Fig 20 are summarized the ones allowed by the European Union.

4-Hexylresorcinol

In what concerns 4-hexylresorcinol its use is still not allowed by the European Union, however the Scientific Committee on Food has assessed the information on the safety of 4-hexylresorcinol and expressed an opinion on March 2003. The committee concluded that 4-hexylresorcinol is toxicologically acceptable for prevention of melanosis in crustaceans, provided residues in crustacean meat do not exceed 2 mg/kg. Based on that information and on the fact that France authorized temporarily the use of the compound under Article 5 of Directive 89/107/EEC it was proposed in October 2004 to the European Parliament the approval of a Directive amending the Directive 95/2/Ec on food additives other than colours and sweeteners and Directive 94/35/EC on sweeteners for use in foodstuffs.

Figure 19 – Maximum levels of SO2 allowed in crustacean according to the Directive 98/72/EC of the European Parliament and of the Council that amends the Directive 95/2/EC on foodadditives other than colours and sweeteners.

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Figure 20 – Sulphites autorized as food additives in the European Union (Directive 95/2/EC on food additives other than colours and sweeteners

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Specifications for chilled and frozen crustacean Product Description Chilled/Frozen Crustaceans Composition Includes crustaceans, prawns, crabs and lobsters Distribution Conditions Distribution temperature:

Chilled Frozen

In ice at -1ºC to 5ºC Minimum of -20°C

Temperature of Seafood Receival and distribution temperature: Chilled Frozen

Minimum -1ºC Maximum 5ºC Minimum of -20°C

Packaging In clean plastic bags inside clean cardboard or insulated containers Labeling Frozen seafood must be labeled with:

> Packer or producer’s name, address and contact phone/fax numbers > Product description: species, size, process method, weight and consumer pack detail > Packing date and/or use-by date >Species name to be in accordance with the EU Directive 2065/2001

Packed-on or Use-by Date Seafood is unacceptable if less than 3 months remains before the use-by date or more than 3 months have elapsed since the packed-on date.

Catch Area According to Council Regulation (EC) nº 104/2000 and Commission Regulation (EC) nº 2005/2001

Heavy Metal Combination Maximum levels permitted (Directive 93/351/EEC, Commission Regulation (EC) nº 221/2002)

Mercury Lead Cadmium Arsenic

0.5ppm 0.5ppm 0.5ppm

Chemical Contamination Polycholorinated Biphenyls Histamine Phosphates Sulfur dioxide and sulphites Fresh, frozen Cooked EDTA Sorbic + benzoic acid Ascorbic acid Formaldehyde

must not exceed 100mg/kg not more than 5 g/kg (Directive 95/2/CE and 96/85/CE) between 150-300 mg/kg 50 mg/kg 75 mg/kg 2000 mg/kg not more than 400mg/kg not more than 50mg/kg

Organic Chemicals Aldrin and Dieldrin BHC Chlordane DDT HCB Heptachlor Lindane

not more than 0.1mg/kg not more than 0.01 mg/kg not more than 0.05mg/kg not more than 1mg/kg not more than 0.1mg/kg not more than 0.05mg/kg not more than 1mg/kg

Microbiological Contamination

Mesophilic aerobic bacteria (30ºC) Escherichia coli (ICMSF) Staphylococci (ICMSF) Salmonella Other pathogens and toxins thereof

m=104, M= 105,, n=5, c=2 not exceeding 10 E. Coli/g not exceeding 103 staphylococci/g free in 25g of food, n=5, c=0 Not present in quantities such as to affect health

Storage Freezer storage temperature: Minimum of -25°C

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Quality control

Apart from better chemical additives and processing techniques, companies involved in crustaceans processing also need, chemical and sensory quality indices for the products classification by levels of quality in order to manage properly the final destination of the catches. On account of this the existence of precise methods for an effective quality control of catches, as well as imported shrimp, is very important and information regarding the acceptable limits for quality parameters fundamental.

Deteriorative changes are the result of several post mortem biochemical alterations and may be perceived by changes in sensorial analysis. Mendes et al. (2005) showed that the levels of biochemical parameters determined in deepwater pink shrimp (Parapenaeus longirostris) at the end of the sensory shelf life at various temperatures were similar and could be considered responsible for the sensory shelf life. Thus, in order to classify a sample of acceptable quality the following limits could be suggested: pH ≤ 7.9, TVB-N ≤ 30 mg/100g, TMA-N ≤ 11 mg/100g, indole ≤ 88 µg/kg, Put ≤ 6 mg/100g, Cad ≤ 7 mg/100g and Agm ≤ 7 mg/100g.

QIM

Quality assurance systems need monitoring of parameters that can be critical throughout the production chain. Freshness and changes in sensory attributes are critical parameters during food production. Information concerning temperature and elapsed time of a catch are of course of major importance. Sensory methods such as the Quality Index Method (QIM) are very useful tools to verify this information. QIM will be useful to give feedback to crew members of fishing vessels concerning the quality of their catch, which may influence better handling on board.

QIM is a quality management tool based upon objective evaluation of certain attributes of raw fish and crustacean (appearance, odor, color, etc) using a points scoring system (from 0 to 3). The scores for all the attributes are then added to give an overall sensory score, the so-called quality index. The quality index increases linearly with keeping time in ice. Therefore the total demerit score can also be used to predict the remaining shelf life. The description of the evaluation of each parameter is written in a guideline. No excessive emphasis is laid on a single attribute so a sample cannot be rejected on the basis of a single criterion. Minor differences in results for any one criterion do not unduly influence the total QIM score. The lower the score the fresher the fish. In Fig 21 is presented an example of a QIM used for Australian prawns and in Fig 22 is displayed a QIM for Norway lobster treated with 4% of a commercial sulphite-based product, and with 4-hexylresorcinol-based formulations, at 0.05% and 0.1% developed in the course of the Project Crustamel CRAFT CT-2002-71517.

.

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Figure 21 - Quality Index Method (QIM) scheme used for Australian prawns

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Quality Parameters Attributes Demerit Points

Odor Fresh 0 Neutral 1 Slightly H3N 2 Off-odor 3 Melanosis Absent 0 Few spots 1 > 25% dark 2 Color in head Natural 0 Yellow/green/grey 1 Eye color Brilliant black 0 Brownish/dully 1 Total 0-7

Figure 22 - Quality Index Method for Norway lobster treated with 4% of a commercial sulphite-

based product, and with 4-hexylresorcinol-based formulations, at 0.05% and 0.1%. (Project Crustamel CRAFT CT-2002-71517).

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Analysis of Residues

Sulphite

Several methods of dosage of sulphites can be used in order to control the respect for the levels imposed by legislation. The most current method is the normalized method of Monier-Williams (Annex 1). This method is easy to set up but the quantification of sulphite is relatively long and needs specific material. Other methods more rapid and practical are available such us the enzymatic method (Annex 2) and the Alert for Sulfites from NeoGen shown in Fig 23 (Annex 3). Though being more convenient the consistence of this method is not yet fully known. In relation to the Neogen method, though its semi-quantitative nature the simplicity is a great advantage.

Figure 23 – Alert for sulphites in seafood from Neogen.

4-Hexylresorcinol

A quantitative liquid chromatography method is used for quantification of 4-hexylresorcinol residues (Annex 4). The method is based on extraction of test portions with methanol followed by liquid chromatographic analysis of the extracts, using a reversed-phase column and fluorimetric detection (excitation: 280 nm, and emission: 310 nm).

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Annex 1: Monier-Williams Method

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Annex 2: Enzymatic Method

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Annex 3: Alert for Sulfites

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Annex 4: 4-Hexylresorcinol

Jonker K.M. and Dekker C.P., 2000. Determination of 4-hexylresorcinol in shrimp by liquid chromatography with fluorescence detection. J. AOAC Int. 2000, 83(1):241-244.

Reagents

(a) 4-Hexylresorcinol - H-6250 (Sigma Chemical Co. St. Louis, MO). Prepare stock solution (5 mg/ml) by weighing 50 mg 4-hexylresorcinol into 10 ml volumetric flask and dissolve in methanol; dilute to volume with methanol. Prepare stock standard (100 µg/ml) by diluting 200 µl stock solution to 10 ml with methanol. Prepare LC stock solution (2.5 µg/ml) by diluting 250 µl stock standard to 10 ml with methanol. Prepare LC working standards (25, 50 and 75 ng/ml) by appropriate dilutions of LC stock solution with mobile phase.

(b) Phosphate buffer (pH 3.0) - Prepare 0.0 1M buffer by dissolving 1.36 g KH2PO4 in 1 l water. Adjust pH with 25% H3PO4 (m/v).

(c) Water ultrapure - Purify through a Milli-Q system (Millipore, Molsheim. France).

(d) Mobile phase - Phosphate buffer— acetonitrile (40+60, v/v).

Apparatus

(a) LC system - Consisting of a Waters Model 625 LC solvent delivery system, a Rheodyne injector, a Jasco Model FP821 fluorimetric detector, and a Millennium 2010 Chromatography Manager (Waters, Milford, MA).

(b) LC analytical column - Reversed-phase C15, 150 x 4.0 mm, 5 µm particle size (Inertsil ODS-2, GL Science, Tokyo. Japan).

(c) Sonifier – MSE Soniprep 150 ultrasonic disintegrator (Sanyo Gallenkamp PCL, Leicester, UK).

Preparation of Extract

(a) Samples - Peel shrimps and homogenize. Add 10 ml water to 5 g homogenate in 50 ml propylene centrifuge tube, and shake tube vigorously for 30 s to disperse shrimp meat in water. Add 15 ml methanol, and homogenize for 30 s with sonifier. Then agitate for 15 min on mechanical shaker at 500 rpm and centrifuge 5 min at 3000 x g. Filter supernatant into 50 ml volumetric flask. Repeat extraction procedure twice, each time with 10 ml methanol, filter, and combine extracts. Wash filter with water, add water to flask, and dilute to volume with water. Shake flask well. Transfer 5.0 ml extract to 20 ml volumetric flask, and add mobile phase to volume. Transfer 1.0 ml of this solution to 1.5 ml Eppendorf vial, and centrifuge for 3 mm at 11 000 x g. Inject 50 µl supernatant into chromatographic system.

(b) Spiked samples - Add 100 µl 4-hexyiresorcinol stock standard (100 µg/ml to 5 g homogenized blank shrimp, representing a 2 µg/g spike. Wait for 15 min. Then prepare extract as described above for samples.

Chromatographic Conditions

Analyze sample extracts by using the following LC conditions: mobile phase flow rate: 0.8 ml/min; injection volume, 50 µl; detector, excitation = 280 nm, emission = 310 nm; run

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time, 10 min. Begin and end each series of analyses with a 4-hexyiresorcinol LC working standard of 50 ng/ml and a spiked sample extract.

Calculations

Calculate recovery (based on 40-fold dilution factor of 2 mg/kg spike, representing an injection level of 50 ng/ml) and concentration of 4-hexyiresorcinol in shrimp as follows:

Recovery, % = Ass / Aws x 100

4-hexylresorcinol, mg/kg = 5/W x 2 x 100/recovery x As / Aws

where Ass = peak area of 4-hexylresorcinol in spiked sample extract; Aws = peak area of 4-hexyiresorcinol in the LC working standard 50 ng/ml; W = shrimp meat weight (g); 2 = content of 4-hexvlresorcinoi 2 mg/kg) represented by the (40-fold dilution) standard solution; and As = peak area of 4-hexylresorcinol in sample extract.

Chromatogram of 4-hexylresorcinol standard (50 ng/ml).

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Annex 5: Quality parameters

Indole method

Reagents

(a) Methanol, HPLC gradient grade (e.g. Merck, Baker) (b) Acetonitrile, HPLC gradient grade(e.g. Baker) (c) Water for HPLC (e.g. Baker, Promochem) (d) Indole (puriss. > 99 %)(e.g. Fluka) (e) 2-Methylindole (purum ~98 %) (e.g. Fluka) (f) Folded filter (diameter: 185 mm) (e.g. Schleicher und Schuell) (g) Fine filter (diameter of pores: 0.45 µm) (e.g. Machery und Nagel)

HPLC Conditions

(a) Column: Use reversed phase column for example Nucleosil 100-5 C18, 250 * 4 mm (b) Guard column: is optional for example Nucleosil 120-7 C18, 11 * 4 mm (c) Detection: Fluorescence detector (for example Shimadzu RF 530 or Thermo Quest

FL3000), Excitation: 280 nm, Emission: 330 nm (d) Injection volume: 20 µl (e) Flow: 0.75 ml/min. (Flow may be adopted.) (f) Mobile phase: Mix acetonitrile and water (for example 550 ml + 450 ml) or methanol and

water (for example 600 ml + 400 ml). Proportion of acetonitrile (or methanol) and water may be adopted to get a sufficient resolution between interfering compounds (at the beginning of the chromatogram), indole, and 2-methylindole.

(g) Retention times: Indole: about 7.5 min. 2-Methylindole: about 9.0 min. It may be useful to wash the column with methanol after a sample series.

Standard Solutions

Indole

I: 1.0 mg/mL Dissolve 100 mg indole in methanol in 100 mL volumetric flask and dilute to volume with methanol. II: 10 µg/mL Pipet 1 mL solution I into 100 mL volumetric flask and dilute to volume with methanol. III: 1.0 µg/mL Pipet 10 mL solution II into 100 mL volumetric flask and dilute to volume with methanol.

2-Methylindole

I: 1.0 mg/mL Dissolve 100 mg 2-methylindole in methanol in 100 mL volumetric flask and dilute to volume with methanol. II: 2.5 µg/mL Pipet 0.5 mL solution I into 200 mL volumetric flask and dilute to volume with methanol.

Calibration Solutions

Prepare calibration solutions indicated in the following table. Pipet indicated volumes into separate 100 mL volumetric flasks and dilute to volume with methanol. Store calibration solutions in refrigerator.

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Calibration Solution

Indole Standard Solution III

(1.0 µg/ml) [ml]

2-Methylindole Standard Solution II

(2.5 µg/ml) [ml]

Water [ml]

2-Methylindole (internal standard)

Concentration [ng/ml]

Indole Concentration

[ng/ml]

1 0.2 1 10 25 2 2 0.5 1 10 25 5 3 1.0 1 10 25 10 4 2.5 1 10 25 25 5 5.0 1 10 25 50

Calibration

(a) Inject 20 µL of each calibration solution. (b) Measure peak heights or areas. (c) Calculate peak ratio R = height indole peak / height 2-methylindole peak

(or area indole peak / area 2-methylindole peak). (d) Calculate amount ratio A = amount indole / amount 2-methylindole (e) Plot R against A. (f) Check if linear or cubic calibration fits best.

Sample preparation

(a) Frozen shrimp: Thaw shrimp (for example in refrigerator during night). Work up shrimp as soon as they are thawed.

(b) Whole shrimp and shell-on shrimp: Withdraw the head and peel shrimp. (c) Frozen shrimp homogenate: Work up homogenate immediately after thawing. It is not

possible to freeze it again for later indole determinations. (d) Homogenate an appropriate amount of shrimp flesh. (e) Weigh 5.0 g ± 0.05 g of shrimp flesh homogenate into beaker. (f) Add 0.5 mL 2-methylindole standard solution II (2.5 µg/mL). (g) Add 45 mL methanol. (h) Homogenate thoroughly with high speed homogenizer for 1-2 minutes. Avoid splattering

/splashing (i) Filtrate the mixture using a folded filter. (j) If filtrate is turbid, filtrate again using a fine filter (diameter of pores: 0.45 µm). (k) Inject 20 µl filtrate into HPLC. (l) Store filtrate in refrigerator. Do not store longer than 2 weeks.

Calculations

Identify peaks of indole and 2-methylindole by their retention times. Calculate peak ratio R and determine amount ratio A from calibration curve. A x b 1000 g µg indole/kg sample = ---------- x ---------- = A x 250 µg/kg c 1 kg A = amount ratio of sample (from calibration curve) b = µg 2-methylindole added to 5 g sample (= 0.5 ml * 2.5 µg/ml= 1.25 µg) c = amount of sample (= 5.0 g)