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close this bookFish Handling, Preservation and Processing in the Tropics: Part 2 (NRI)
View the document(introduction...)
View the documentSummaries
View the documentAcknowledgements
View the documentIntroduction
View the documentSalting of fish: salt
View the documentSalting of fish: methods
View the documentDrying of fish: basic principles
View the documentDrying of fish: methods
View the documentSmoking of fish
View the documentMarinades
View the documentFermented fish products: a review
View the documentBoiled fish products
View the documentFish canning: theory and practice
View the documentFreeze drying
View the documentIrradiation
View the documentMiscellaneous products: crustaceans
View the documentMiscellaneous aquatic products used as food
View the documentFood by-products
View the documentNon-food by-products
View the documentNew and delicatessen products
View the documentFish meal
View the documentFish silage
View the documentChemical and physical methods of quality assessment
View the documentOrganoleptic (sensory) measurement of spoilage
View the documentMicrobiology of spoilage
View the documentMicrobiology of fish spoilage
View the documentPublic health microbiology
View the documentInternational standards for fisheries products
View the documentLarge-scale fish landing facilities
View the documentSmall-scale landing facilities: design and operation
View the documentRetail sale facilities
View the documentFisheries extension services: their role in rural development
View the documentTraining in the field
View the documentAppendix

Chemical and physical methods of quality assessment

Attributes of the hypothetical ideal method

Over the years many different methods have been developed and investigated in an attempt to find the most suitable index for use in quality control testing. The 'ideal' method would have the following attributes:

(a) It would be non-destructive.

(b) The equipment would be cheap to purchase and maintain.

(c) The equipment would be robust, portable and simple to operate and suited to minimum facility 'field' operations.

(d) It would produce consistent results on standard material.

Few methods even approach this ideal.

Precautions applicable to any quality assessment operation

(a) Ensure that your sample of fish from the bulk truly represents the bulk.

(b) Ensure that, when a part of a fish is analysed, it is always the same part. As discussed previously, composition differs markedly from head to tail of true fish, and depends particularly upon the relative amounts of skin, dark muscle and light muscle that are in the sample.

(c) Ensure that a rigidly standardised method is employed. The result obtained frequently depends upon the analytical method employed. Accordingly, standards specifying grades related to the content of a particular component should also specify the method of obtaining the result.

(d) Never attempt to compare directly results obtained by different methods.

(e) In export operations, levels of acceptance and rejection must correspond to the importer's legislation and/or specifications.

(f) In local operations, levels of acceptance and rejection should be related to local requirements.

Chemical methods

(a) Trimethylamine (TMA)

Trimethylamine, N(CH3)3, smells like ammonia and is chemically similar to ammonia, NH3. It is produced by many spoilage micro-organisms from a compound known as trimethylamine oxide (TMAO), O=N(CH3)3.

This conversion can only occur if:

(i) TMAO is present
(ii) suitable spoilage micro-organisms are present.

TMAO is not normally present in freshwater fish but is found in marine species at a level related to the salinity of the habitat. The level of TMAO may change as a fish migrates from water of one salinity to another. High salinity is associated with high TMAO contents. It has been observed that freshwater fish may contain TMAO if they have been fed with fish meal made from marine species.

Table 1 Variations in TMAO content (mg N/100g) in Indian prawns: effect of fishing ground salinity

If TMAO is present in the muscle, then micro-organisms will normally invade postmortem and produce TMA, slowly at first, then with increasing rapidity in fish stored at ambient temperature, in ice or in refrigerated seawater.

Careful processing, e.g. heavy salting, salting and hot smoking, drying thoroughly, freezing, canning etc., either destroys or inhibits the TMA producing microorganisms and any TMA in such products was probably already present at the time of processing.
If fish are stored or processed in contact with water or melt-water, some TMA will be washed out, masking the degree of spoilage. This occurrence is most pronounced in small prawns which have a large surface area in relation to volume.

Table 2 The leaching of TMA from prawns (Xiphopenaeus croyeri) stored in ice

Measurements of TMA are often used to assess the quality of fresh and frozen fish but the result obtained depends upon the method used: rigid standardisation is essential and it is unsound to make direct comparisons of results obtained by different methods. All methods require an acidic extract of the fish muscle and it is usually necessary to prevent interference by ammonia.

Simple methods of analysis:

(i) Conway microdiffusion or steam distillation. This method is relatively simple and uses relatively cheap equipment.

(ii) Trimethylamine sensitive electrode and pH meter. The assembly of equipment for this method is not yet commercially available; the capital cost probably approaches £1 000 but the equipment is cheap and simple to operate; it is essentially portable but a power supply is required; it is promising for the future.

More sophisticated methods of analysis:

(i) Measurement of the colour produced by treating TMA with picric acid. This method requires a colorimeter and mechanical sample shaking equipment, and a practised and conscientious operator.

(ii) Gas liquid chromatography. This method requires sophisticated equipment and a skilled operator; ammonia does not interfere.

Many workers feel that TMA content has only a poor correlation with eating quality, particularly in the early stages of spoilage. Typically, the presence of more than a trace is taken to indicate that spoilage has already occurred. It is not possible to use the TMA content to predict the remaining useful storage life.

(b) Total volatile nitrogen (TVN)

The abbreviation TVN refers to any volatile nitrogen-containing compounds that are produced post-mortem. The main components are TMA and ammonia. In a few species, dimethylamine is also produced e.g. Japanese hake (Lutella sp.). The relative amounts of these compounds depend upon the type of fish being examined and its quality. The following figures may be considered typical:

1. Freshwater fish - almost entirely ammonia.
2. Bony marine fish - ammonia equalling or slightly exceeding the TMA.
3. Cartilaginous marine fish - ammonia usually markedly exceeding the TMA.
4. Crustacean shellfish - ammonia usually more extensive than in bony marine fish but not as extensive as in cartilaginous marine fish.

TMA has already been fully discussed. The ammonia is mainly produced by bacterial attack on proteins and also by attack on amino acids (particularly arginine in crustacea) and on urea in cartilaginous species.

TVN determinations can be made by any of the methods mentioned for TMA but without the need to prevent interference by ammonia.

(c) Nucleotide degradation products

Adenosine triphosphate (ATP) is the major nucleotide of living muscle. Post-mortem, it is degraded by enzymes that are naturally present in the muscle and probably by micro-organisms which have invaded the flesh. It is generally accepted that a measure of nucleotide breakdown is more closely related to eating quality than a measure of TMA or TVN, especially in the early stages of spoilage.

A full discussion of ATP breakdown is beyond the scope of this course, but the possible pathways are summarised below:


In crustacean species which are cooked alive, the degradation probably does not proceed beyond adenosine monophosphate. In a few species, degradation may occur via adenosine but, in the majority of species of commercial importance, degradation occurs via inosine monophosphate. The rate at which this degradation occurs is variable with species and with season, being markedly influenced by the pH value of the flesh. It is also influenced by temperature, occurring more rapidly above ambient temperature than in frozen storage. In most species, the final product is hypoxanthine but Japanese workers have reported that, in many Pacific species of commercial importance, inosine may be the final product (See Table 3).

Inosine monophosphate is considered to be a desirable component contributing to the characteristic flavour of fresh fish. In contrast, hypoxanthine is said to have an undesirable bitter taste. Opinion is divided about the taste of inosine but it is clear that nucleotide degradation involves at least a loss of a desirable component (IMP) and, in many cases, the accumulation of an undesirable component (Hx).

Hypoxanthine is not very soluble in water and so is not easily leached, as are TVN and TMA. It is essentially stable during processing and its presence in canned fish is indicative of the pre-processing quality.

It has been observed that, for a given species, the rate of accumulation of Hx is proportional to the temperature of storage. If this rate is known, and the Hx concentration corresponding to the reject limit is also known, then the remaining useful storage life can be estimated by measuring the Hx content.

Simple methods of analysis:

(i) The simplest method is to use test papers that are dipped in an aqueous extract of fish and to compare the colour so generated with preestablished standards. The test papers contain the enzyme xanthine oxidase and the dyestuff dichlorophenolindophenol (DCPIP). This enzyme converts hypoxanthine to uric acid, at the same time bleaching the pink DCPIP. At present, these papers are not commercially available but may be prepared in a moderately equipped laboratory. They are stable if kept dry, and easily portable.

(ii) The older enzymic methods require more sophisticated equipment and use acidic protein free extracts. A colorimeter, or UV spectrophotometer, is required.

More sophisticated methods of analysis:

(i) Hypoxanthine may be precipitated by silver or barium salts and the precipitate recovered and weighed. This method is essentially simple but requires accurate balances and very careful operators.

(ii) Ion exchange resins may be used to separate individual nucleotides, and their degradation products, but this method is not suited to the analysis of a large number of samples. A modification is possible, permitting a separation of the degradation products into two groups: those containing phosphate and those which do not. The ratio of phosphate-free to phosphate-containing products may be used to calculate the K value. It is claimed by Japanese workers to be more useful than a Hx value for those species where degradation stops at inosine (See Table 3).

Table 3 Pacific fish species known to yield inosine as the major nucleotide degradation product

(d) Fat degradation products

Whether a fish contains a little or a large amount of fat, the degradation of this fat during storage can cause undesirable changes in flavour, odour and texture. Unfortunately, fat degradation (or rancidity) is only conveniently measured on extracted oils. Therefore, it is necessary to extract the oil from the flesh first. In practice, this means that such tests can only be applied to those fish of high fat content.

The standard analyses of fat rancidity appear to be simple but, in practice, fat extraction and/or fat analysis may alter the composition to such an extent that the results are meaningless. Such methods are much more appropriate for assessing the quality of extracted oils obtained as commercial by-products.

The routine analyses are:
(i) Free fatty acids content, which is a titration.
(ii) Peroxide value, which must be performed in subdued light, and is a titration.
(iii) Aldehyde detecting reactions, which are colorimetric.

(e) Salt and moisture content

The quality of products such as salted fish, sun dried fish and smoked fish is essentially determined by:

1. The quality of the fish before processing.
2. The adequacy of the processing.
3. The adequacy of the storage.

The tests previously referred to may be applied to the fish before processing. If good quality fish are used and they are carefully processed and properly stored, then a good quality product will be obtained. The products are preserved primarily by reducing the water activity, i.e. by reducing the water content and/or increasing the salt content; analyses of these components serve as a check on the processing.

Moisture analysis:

Take an accurately weighed sample and cut into small pieces. Dry in an oven at 105°C for 3 hours. Break up the pieces, taking care not to lose any sample, and dry to constant weight. The weight loss is taken as the water content.

Salt analysis:

Take a small sample and homogenise in distilled water to thoroughly extract the salt. Centrifuge and dilute the supernatant to volume. Determine salt by titration with silver nitrate. The volume of silver nitrate used is proportional to the salt content.

Chloride meters are available but their performance may be erratic when used on protein-rich extracts.

Physical methods

(a) Measurements of flesh impedance and capacitance

The term impedance may, in practical terms, be considered as resistance to alternating current. (In fact that is an over-simplification and it is more accurate to refer to resistance and inductance.) The term capacitance may be considered as a measure of the ability to retain electrical charge.

Instruments capable of measuring either impedance, or impedance and capacitance, have been developed over the last 30 years. The most recent and most satisfactory instrument is known as the Torry Fish Freshness Meter (TFM). The TFM has four electrodes which are placed upon the fish. It is most important that a consistent position is used for all measurements. Two electrodes measure the impedance and capacitance; the other pair ensure good electrical contact and automatically correct the reading to the value which would be observed at 0°C. The reading is displayed digitally in the range 1 to 19; rarely does the value exceed 16 for UK fish, such high values corresponding to the highest quality fish.

The rate at which the TFM reading declines depends upon the species. It is known that changes in proteins and cell membranes caused by enzymes and microorganisms are responsible for the fall in the TFM reading as the fish deteriorate. Physical damage caused by rough handling and bruising also markedly reduces the TFM reading, probably to a greater extent than the physical damage reduces the eating quality, and many workers consider this a disadvantage. When applied to fatty fish, the TFM reading is also markedly influenced by the fat content (and thus the season) and, although the fat content may genuinely influence the eating quality, this fact is also looked upon as a disadvantage. Since the properties of the flesh and skin are different, fish with the skin on show different results from fish that have had the skin removed.

Current cost of the TFM is in the region of £400. However, it is easy to use, robust, portable and ideally suited to field operations. The only routine maintenance is to recharge the batteries daily. So far, this instrument has been little used in the tropics but UK experience suggests it could be of some value.


With reference to the hypothetical ideal quality assessment method mentioned at the beginning of this lecture, the chemical and physical methods most closely approaching the ideal are:

(i) the xanthine oxidase impregnated test paper;
(ii) the Torry Fish Freshness Meter;
(iii) a trimethylamine or total volatile nitrogen sensitive electrode system;
(iv) the Conway microdiffusion method for TVN.


For Trimethylamine (TMA) Method

1. CASTRO, L. A. B. (1975) Boletim do Instituto de Pesca, 4, (2), 29 - 36.

2. CHANG, G. W., CHANG, W. L. and LEW, K. B. K. (1976) Journal of Food Science, 41, 723 - 724.

3. MONTGOMERY, W. A., SIDHU, G. S. and VALE, G. L. (1970) Council for Scientific and Industrial Research Food Preservation Quarterly, 30 (2), 21 - 27.

4. RUITER, A. (1971) Voedingsmiddelentechnologie, 2, 1 - 10.

5. VELANKAR, N. K. and GOVINDAN, T. K. (1960) Proceedings of the Indian Academy of Science, 52B, pp. 111 - 115.

For Total Volatile Nitrogen (TVN) Method

1. BURT, J. R. (1977) Process Biochemistry, 12 (1), 32 - 35.

2. CLIFFORD, M. N. and KUMAR, N. Grimsby College of Technology (Unpublished results).

3. ESHIRA, S. and UCHIYAMA, H. (1975) Bulletin of the Tokai Regional Fisheries Research Laboratory, No.75, p. 63.

4. JAHNS, F. D., HOWE, J. L., CODURI, J. R. and RAND, A. G. (1976) Food Technology (USA), 30 (7), 27 - 30.

For Physical (TFM) Method

CHEYNE, A. (1975) Fishing News International, 14, (12), 71 - 76.