Radurization of Scampi, Shrimp and Cod

Home , Shrimp and prawn as food, Stockfish

TECHNICAL REPORTS SERIES No. 124

JOINT FAO/IAEA DIVISION OF ATOMIC ENERGY IN FOOD AND AGRICULTURE ié)

INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1971

RADURIZATION OF SCAMPI, SHRIMP AND COD

TECHNICAL REPORTS SERIES No. 124

RADURIZATION OF SCAMPI, SHRIMP AND COD

by G. HANNESSON and B. DAGBJARTSSON

Icelandic Fisheries Laboratories Reykjavik, Iceland

REPORT OF A PROJECT ORGANIZED AND SUPERVISED BY THE JOINT F AO/IAEA DIVISION OF ATOMIC ENERGY IN FOOD AND AGRICULTURE

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1971 RADURIZATION OF SCAMPI, SHRIMP AND COD IAEA, VIENNA, 1971 STI/DOC/lO/124

Printed by the IAEA in Austria April 1971 FOREWORD

In January 1968 an agreement was reached in Vienna by representatives of the Governments of the United States of America, Iceland and the Inter- national Atomic Energy Agency (IAEA) to undertake a survey project on the use of ionizing radiation for extending the storage life of seafoods. The United States Atomic Energy Commission (USAEC) provided an irradiator for a period of eighteen months. The irradiator, originally used on the US Bureau of Fisheries exploratory vessel M.V. Delaware, was furnished with a new cobalt-60 radiation source at the Brookhaven National Laboratories (BNL). It was assembled and installed on the ground floor of the Icelandic Fisheries Laboratories (IFL), Reykjavik, under the supervision of Mr. B. Sylvester of BNL. The activity of the source was approximately 35 000 Ci. The project was started in July 1968. The IFL furnished laboratory facilities, rawmaterial, manpower and other services. The IAEA granted fellowships for training two scientific staff members of the IFL in the irradiation of foods and the operation of irradiation facilities. It was also agreed that the IFL would accept and provide facilities for IAEA Fellowship holders, from various countries, who would participate in the project as scientific co-workers. The average stay was for two months. The USAEC provided a fisheries irradiation expert, Mr. J.D. Kaylor of the Bureau of Commercial Fisheries, Gloucester, Mass., who stayed at the IFL from 16 June to 16 July 1968. Under his supervision the ground- work was laid for satisfactory radiation safety procedures in co-operation with Dr. G. Jónsson, Director of the Icelandic Radiological Health Labora- tory. Mr. Kaylor was also occupied in training and familiarizing the scientific staff members in radiation calibration and dose determinations. The main objective of the survey was to demonstrate whether radiation preservation is practical under the type of conditions that exist in the fishing industry and trade in Iceland. As the domestic market in Iceland is very limited, it is important to export as much of the seafood catch as possible. Most of the catch is salted, dried or frozen. Nevertheless a considerable quantity, amounting to 30 000 - 40 000 tonnes, of iced fish such as whitefish (e.g. cod and haddock) and herring is shipped annually to the United Kingdom and Continental markets, where it is sold in the fresh state. Because some of this iced fish arrives in an unsaleable condition, it seemed desirable to determine whether the storage life of fresh Icelandic seafoods could be extended by radiation preservation beyond what is attained by icing. No attempts as yet have been made to market fresh, iced lobster, shrimp and other shellfish because of lack of satisfactory preservation techniques. The investigation was concentrated on one species of fish, cod, and two crustacean species, scampi (Norway lobster tails) and deep-sea shrimp. The results showed that the storage life of cod fillets at 0°-l°C, irradiated at a dose level of 2 X 105 rad, could be extended by a factor of about 3, namely up to 19 days for trawl cod and 24 days for line cod. The market life of blanched shrimp and scampi, irradiated at a dose level of 1 X 105 to 2 X 105, could be increased to about 6 weeks when stored at 0°-l°C; this is about twice the market life for unirradiated specimens. Clearly these results could have commercial importance. The authors of this report express their gratitude to the organizations who enabled the work to be undertaken, and to the many scientists who participated in the project. CONTENTS

PART I. RADURIZATION OF NORWAY LOBSTER TAILS (SCAMPI) 1

1. INTRODUCTION : 1

2. MATERIALS AND METHODS 2 2.1. Processing methods 2 2.2. Analytical methods for storage life determination 3

3. RESULTS 5 3.1. Results of preliminary experiments 5 3.2. The effect of processing treatments on number of surviving micro-organisms (bactericidal efficacy) 27 3.3. Results of storage tests at 0° - 1°C and 5° - 6°C on the effect of various treatments on the total bacterial count (TBC), sensory evaluation (T-tests) and volatile acid number (VAN) 27

4. DISCUSSION 29 4.1. The radiation dose 29 4.2. Sensory evaluation (T-test) 29 4.3. Volatile acid number (VAN) 30 4.4. Bacteriological aspect of the study 31 4.5. Comparison of factors influencing storage life 32 4.6. Possible use of irradiation for processing and preservation of lobster 33

5. SUMMARY AND CONCLUSIONS 34

PART II. RADURIZATION OF DEEP-SEA SHRIMP 35

6. INTRODUCTION 35

7. MATERIALS AND METHODS 35 7.1. Materials 35 7.2. Methods 43

8. RESULTS 46 8.1. Bacteriological determination (TBC) 46 8.2. The effect of various processing treatments and irradiation on storage life 53 9. DISCUSSION . 56

10. SUMMARY AND CONCLUSIONS 59

PART III. RADURIZATION OF WHOLE COD AND COD FILLETS.. 61

11. INTRODUCTION 61

12. MATERIALS AND METHODS 62 12.1. Raw material, origin and preparation 62 12.2. Packaging 63 12.3. Storage 63 12.4. Irradiation dose 63 12.5. Sensory evaluation (O-test) 64 12.6. Chemical tests 65 12.7. Bacteriological determination (TBC) 65

13. RESULTS 66 13.1. Results of O-test 66 13.2. Results of chemical tests (VAN and TMA) 69 13.3. Bacteriological determination (TBC) 82

14. DISCUSSION 86

15. SUMMARY AND CONCLUSIONS 90

ACKNOWLEDGEMENTS 92 REFERENCES 92 PART I RADURIZATION OF NORWAY LOBSTER TAILS (SCAMPI)

1. INTRODUCTION

The project began in the midsummer of 1968 after installation, testing and calibration of the irradiator [1] . At that time of the year Icelandic fisheries are based mainly on whitefish and herring. However, this is also the high season for lobster. The lobster season lasts from June until September. The main whitefish season (cod, haddock, etc.) is from January until May. It was therefore felt logical to start the irradiation project with the lobster, which was readily available. The species of lobster which lives in the waters off Iceland is the Norway or Dublin lobster, whose scientific name is Nephros norvegicus. Norway lobsters and particularly the shelled tail-meats have become known as scampi. The lobster is found in the Faeroes and off Iceland, it ranges from the coasts of Norway, Scotland and Ireland to the Mediterranean. The lobster is fished commercially in Iceland using rather small boats by means of a specially designed light lobster trawl. On board the boats the head and carapace, with claws and legs attached, is twisted off and dis- carded leaving only the unshelled tails to be iced until landed. The amount of meat in the claws is very small and at present is not salvaged. Also the separation is carried out because the digestive glands in the dead, whole lobster would rapidly break down the edible flesh. The use of refrigerated seawater for preservation of lobster tails has not given good results, primarily because of difficulty in controlling the proper ratio of ice to seawater. The iced lobster tails are processed as soon as possible, i.e. washed, degutted by a mechanical device, graded as to size, wrapped in parchment and packed in 5-lb wax boxes, and frozen in plate freezers. This product is the highest priced and most sought after. Deformed, broken and dis- coloured tails are panfrozen to be deshelled during off-season, and fetch a substantially lower price. Lobster fishing is a new branch of the Icelandic fishing industry and is localized on the southwest coast. The mean annual catch during 1960-66 was about 3000 metric tons of whole lobster. Prime lobster tails are sold wholesale for $3.55 per kg after packaging and freezing. Compared with other seafood products the price is very high and obviously it is essential that product losses be kept at the absolute minimum. Lobsters [2] have a relatively short storage life compared with other iced fish products. The chief spoilage patterns can be characterized as follows: (1) black colour (melanosis) formation, also known as "black spot"; (2) rapid and high ammonia and trimethylamine (TMA) production; and (3) microbial spoilage [3] . Black colour formation is caused by auto-oxidation of certain chromo- genes of the phenolamine type [4] which in turn are caused by enzymatic breakdown of proteins.

1 Measures to prevent the formation of black colour include both chemical and physical means. Chemicals such,as ascorbic, citric and tartaric acids, sulphites and ordinary salt have been used as dips or incorporated in ice[3,4] . One of the most effective and simple ways to prevent melanosis is by blanching in boiling water. By blanching is implied dipping the product in boiling water for a specified short time. The term precooking is also used in this connotation, particularly for shrimp. No significant data are available which account for deteriorative changes causing spoilage in raw lobster. There are, however, several indications that they follow the general pattern for crustaceans [3, 5] . Like other crustaceans, lobsters are characterized by a relatively high content of mono-aminonitrogen. The breakdown through autolysis and bacterial de- composition primarily attacks the protein and results in a relative decline of protein content and increase in volatile nitrogenous compounds and fatty acids (volatile basis, trimethylamine, volatile acids, etc.). As indicated above, frozen lobster tail is a highly priced product. The purpose of the project was to find out whether ionizing radiation could fit in to existing processing methods in Iceland, whereby it would be possible to extend the storage life of the fresh product so that it could be shipped to foreign markets. Iced lobster tails cannot be exported because of limited storage life. It became apparent shortly after starting the irradiation experiments on lobster that ionizing radiation alone would not prevent the enzymatic changes (melanosis) in the product. Experiments done earlier in the Iceland laboratory (unpublished data) on various shellfish species had given promising results in preventing melanosis by blanching. Preliminary tests done in the early phase of this project substantiated this observation.

2. MATERIALS AND METHODS

2.1. Processing methods

2.1.1. Raw material

The lobster tails used for the experiments were obtained mainly from two commercial lobster freezing plants located in the vicinity of Reykjavik. The lobster tails were taken off the processing line after washing, degutting and grading, and at this stage were ready for packaging and freezing. In all instances the lobsters were recently landed but most of them had been one to three days out of the sea. As will be evident later in Parti, this resulted in raw material of varying quality. The lobster tails were transported under ice directly to the laboratory where they were processed the same day. The total amount of lobster tails used in all the experiments was about 135 kg.

2.1.2. Packaging

At the beginning of the experiment the use of thick plastic packaging film (up to 0. 1 mm) was tried without success, as the spines of the ventral side and the tailfin segments tore the pouches. It was then decided to use Metal Box metal cans (no. 400 X 212, i.e. 4 in. inside diameter by 2 12/16 in. high) which hold about 1/2 kg or 500 cm3). Obviously metal cans would not be used commercially. Some types of plastic packaging film or aluminium

2 foil would be the packaging materials of choice in the trade. On the average about 300 grams of product was placed in each can. The cans were then hermetically sealed in a can closing machine.

2.1.3. Irradiation procedure

Dosimetry was performed before the experiments began to determine the dose rate for lobsters in metal cans [6] . A vial of Fe/Cu solution was placed in the centre of each can, which was then filled with lobster tails. The cans, 11 in all, were placed in the irradiator carriers (containers). In order to get an even dose distribution, the empty space between the cans was filled with rice. The maximum/minimum dose ratio within each can was found to be between 1.10-1.15 (or 10-15% difference), but the accepted dose rate was calculated from the dose which the centre of the cans received. The difference in dose rate between the centre position of the can which received the highest dose and that which received the lowest (the lower corners of the carrier) amounted to a variation of 25%. An average dose rate of 435-445 rad/sec was found. Doses of 1 X 105 , 2 X 105 and 3 X 105 rad were used in these experiments.

2.1.4. Blanching

The reasons for blanching the raw material have been stated above [3] . Blanching was performed as follows: about 1. 5 kg of lobster tails were placed in a wide mesh wire-basket and dipped into about 5 litres of boiling water. The temperature of the boiling water was thereby lowered to 85-90°C. After a specified time interval the basket was removed from the hot water and cooled in running cold tap-water for the same length of time as it was immersed in the hot water (equivalent to blanching time). The contents of the basket were then drained and placed in metal cans which were mechani- cally sealed. Blanching time trials of 1/2, 1 and 2 min showed that 1/2 min blanching was insufficient both alone and in combination with radiation. Therefore in the experiments, where the raw material was either blanched only or blanched and irradiated, the blanching times used were 1 or 2 min.

2.1.5. Storage

As soon as the cans had received the predetermined treatment (blanching, irradiation, or neither), they were placed in cold storage rooms. The storage temperatures chosen wereO°-l°C and5°-6°C. The cans were stored and periodically tested until the quality criteria used indicated termination of each experiment. Samples were removed from the storage room at the time of testing in order to prevent any temperature increase or variation.

2.2. Analytical methods for storage life determination

2.2.1. Sensory evaluation (T-test)

The method of sensory evaluation [7] is based on a five-point scale (5 = very good; 4 = good; 3 = fair; 2 = borderline and 1 = poor or inedible)

3 as judged by an expert group of five to seven panel members. Odour and flavour were scored, but appearance was not tested as this depended entirely on whether the lobster had been blanched or not (see results and discussion below). The preparation of the samples for sensory evaluation was as follows: about 8-10 lobster tail pieces from the same can were placed in a minimum amount of boiling water without salt or any other additives. After the water started to boil again, boiling was continued for 7 min. The containers were then removed from the heat source and placed in front of the panel member. Each container carried a code number, unknown to the panel member. Care was taken to ensure that the panel member judged the product independently. Scores were recorded on specific forms. At least one sample of each batch being subjected to sensory evaluation was of good quality. Quick frozen reference lobster tails were used if the test material was of inferior quality. The maximum number of samples tested at one time never exceeded four. 2.2.2. Volatile acid number (VAN)

According to Spinelli et al. [8] , the volatile acid number determination is the most indicative chemical method for quality assessment of irradiated seafoods. The method used was that of the AOAC [9] with minor modifications. Fifty grams of lobster together with 180 ml of water was mixed in a Waring blender for 1 min. Twenty-five ml of IN H2S04 and 40 ml of 20% phosphotungstic acid was added and mixing resumed for 1/2 min. One hundred and fifty ml of the slurry was then filtered and made acid to congo red indicator, introduced to steam distillation apparatus and distilled until 200 ml had been collected. It was then titrated with 0.01N NaOH to phenolth- phalein end point. The ml of NaOH minus the blank multiplied by 4 gave the VAN value. 2.2.3. Bacteriological determinations (TBC)

A description of the sample preparation and radiation treatment has been given above. It should be pointed out that the samples used for bacterial counts were taken aseptically from a sample can, which subsequently was used for other determinations, i.e. sensory testing and volatile acid number determinations. This manner of sampling was necessary as the raw material (lobster tails) is very expensive and all efforts were made to economize. In detail, sampling and bacterial counts were made in the following way: a portion of lobster tails weighing 44 g was removed from the can and transferred to a Waring blender jar, and 400 ml of physiological saline solution added. The contents was then homogenized for min at high speed; the slurry was then allowed to settle for 5 min. Serial decimal dilutions were prepared in physiological saline solution and total viable counts made by pour plate method using modified TPY agar [10] . The modified TPY agar contains trypticase, 15 g; phytone, 5 g; yeast extract, 5 g; NaCl, 5 g; dextrose 2.5 g; agar, 15 g; distilled water, 1000 ml; pH 7.2. Incubation temperature was at 22°C for 72 h and count- ing was done with the aid of a Quebec colony counter. For further details on choice of media and incubation temperature, see the results and discussion below.

4 3. RESULTS The results of experiments carried out in this study, except for preliminary experiments which are discussed in Section 3 (3.1 - 3.3) are to be found in Tables I - VIII and Figs 1-7. The two main criteria used for determining the end of storage life or the borderline of acceptability are the total bacterial counts of 1 X 106 per gram at incubation temperature of 22° С and a sensory evaluation score of 2 on a 5-point scale test. In addition volatile acid determination (VAN) was attempted for the same purpose, and the results and limitations of this method are discussed in Section 4.3.

3.1. Results of preliminary experiments

3.1.1. Experiments on dipping and blanching

As mentioned above, irradiation does not prevent black colour formation (melanosis) in the lobster tails. As a matter of fact, irradiation seems to enhance its formation. A noticeable difference could be detected, after storage, between irradiation doses of 1 X 105 and 3X 105 rad as blackening of the lobster tails was more pronounced at the latter dose. Furthermore, after a time in storage, the black colour of the lobster tails receiving irradiation appeared to be more distinct than that of the unirradiated product. Other means of preventing melanosis had to be employed. Two alter- natives existed, chemical and physical treatment [ 3, 4] as mentioned above. As a chemical treatment a dipping solution containing 0.45% citric and 0.05% ascorbic acid was made. The product was dipped in this solution for 10 min and drained. A portion of the dipped batch was irradiated (2 X 105 rad), but the remainder kept as reference material. After two days storage at 0° - 1°C the irradiated product had definitely darkened to such an extent that its appearance was judged unacceptable. The following day the unirradiated product was judged unacceptable for the same reason. It should be noted, however, that the quality of the raw material was questionable. No further trials using chemical dips were done. The physical treatment involved application of heat, the heat treatment used in this study being blanching in boiling water (see Section 2.1.4). Blanching alone for \ min showed inconsistent results. For example, great variation between samples of the same treatment was noticed, as judged by appearance (melanosis) as well as TBC, T-test and VAN. It was, therefore, felt that blanching alone for | min appeared to be an unreliable treatment for obtaining extended storage life of the product. This can be further substantiated by the low bactericidal effect of the treatment (see Fig.7). However, subsequent irradiation of the product blanched for | min showed a good bactericidal effect on the microflora and consequently extended storage life, but darkening of the product was not prevented. Further trials using blanching for | min were not done. The inconsistent results may be accounted for by uneven heat penetration. Preliminary trials also included longer blanching times, i.e. 1 and 2 min. Results of those experiments are shown in Tables I - VIII and Figs 1-7, and are discussed in Section 3.3 and Section 4. Blanching causes changes in the physical appearance of the lobster tails.

Text continued on page 26

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12 TABLE VIII. THE STORAGE LIFE OF LOBSTER TAILS AFTER VARIOUS TREATMENTS AND STORAGE TEMPERATURES AS JUDGED BY THE BORDERLINE OF ACCEPTABILITY FOR THE TOTAL BACTERIAL COUNT (TBC) ë IX 106, THE SENSORY EVALUATION SCORE (T-TEST) S 2, AND VOLATILE ACID NUMBER (VAN) AT END OF THE EXPERIMENTS

TBC г Dose T-test £2 VAN Temp, of storage Treatment 1 x 10s after (rad) after daysa (at end of experiment) days3

0° - 1°C Without blanching 0 1-6 5-6 40 1 X 105 11-13 12-18 17 " 2 X 10 s 14 18 12 3 X 10s 24-25 20 10 Blanched 1 min 0 6-8 12-13 79 1 X 10 5 15 25 16 2 X 10 s 18 29 14 " 3 X 10s 29 30 11 Blanched 2 min 0 9-13 20-22 12 1 X 10s 19-26 39 18 2 X 10s 23 40 8 " 3 X 105 42 45 11

5° - 6°C Without blanching 0 1 2 136 " 1 X 105 4 4 20 2 X 10s 6 6 12 3 X 105 9 9 22 Blanched 1 min 0 3-5 5 60 1 X 10s 8 13 17 2 X 10s 10 15 12 3 X 10s 10 17 14 Blanched 2 min 0 5-7 7 9 1 X 10s 8-15 13 12 2 X 10s 14 18 11 3 X 105 19 21 7

a Results given as a range signify values obtained in more than one experiment.

13 Doy»

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FIG, 3. Comparison of the effect of different dose level radiation of lobster tails on total bacterial count (TBC = — О — ), sensory evaluation (T-test score = — e — ) and volatile acid number (VAN = — О — ). Blanching time 1 min. Storage temperature 0°-l°C.

Oays

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25 KILLING EFFICACY н Log of number of ra Log of number of bacteria Q nufT1ber of bacteria surviving bacteria killed by irradiation killed by blanching

Blanching followed by irradiation

Processing treatment FIG. 7. The number of bacteria surviving various processing treatments.

The shell becomes lighter in colour and the exposed meat surface congeals and takes on the appearance of a cooked product. The longer the blanching time the more distinct are the changes in appearance.

3.1.2. Trials on maintaining the product at various temperatures during storage and shipment The conventional storage temperature for fresh seafoods is from 0° - 1°C (iced product) and up to 6°C (refrigeration). Consequently, these temperatures were used for these storage studies. In commerce, however, it is inevitable that product temperature may at times reach the ambient or higher temperatures. In order to check this point samples of blanched and irradiated product were stored at room temperature and body temperature. Within 24 hours all the samples were definitely spoilt. To simulate actual conditions under transport to potential foreign markets, two shipments were made to Vienna in co-operation with the IAEA. The first shipment was by surface (by sea and by rail). The product was canned and packed with ice in suitable containers. The shipment was re-iced once under way and was examined in Vienna 13 days after processing, having been for an unknown length of time at ambient temperature. According to comments received from IAEA experts, samples which had received 3X 105 rad without blanching were judged to be of fair quality. The second shipment to Vienna was made by air transport without ice. This involved a product which had been blanched for 2 min and had received a dose of 1 X 105 rad. Total elapsed time to inspection is unknown, but probably amounted to 6 or 7 days. From the comments received it is gathered that at least some of the shipment was equal to a frozen reference product.

26 3.2. The effect of processing treatments on number of surviving micro-organisms (bactericidal efficacy)

The bactericidal efficacy of a given dose of irradiation depends on the kind and species of micro-organisms, the number and condition of micro-organisms originally present, the composition of the food and the physical state of the food during irradiation [11]. In this study the bactericidal efficacy of pasteurization doses of irradiation and heat treatment (blanching) was determined either as a single factor or in combination. The results are shown in Tables I - VIII and Fig.7. The bactericidal effect of the two processing treatments may be seen most clearly in Fig.7 where various individual treatments and combined treatments are compared with respect to the logarithm of the initial and surviving number of organisms. The average reduction of bacterial numbers when blanching treatment alone was used is as follows: (1) blanching for \ min reduced the bacterial number by 1.2 log cycles; (2) blanching for 1 min, 2.0 log cycles; and (3) blanching for 2 min, about 2.5 log cycles. In evaluating the lethal effect of the various blanching times it may be observed, firstly, that increased blanching time appears to increase the overall efficacy of killing micro-organisms and secondly, that apparent variation exists within each blanching time experiment of 2 min. Although in- conclusive, the killing efficacy seems to depend on the initial number of micro-organisms. The exceptions seen in trials with 2-min blanching could be explained on the basis of localization of micro-organisms in the lobster. The average reduction of bacterial numbers when irradiation treatment only was applied was as follows: (1) a dose of 1 X 105 rad reduced the bacterial number by 2.2 log cycles; (2) a dose of 2 X 105 rad reduced the number by 3.3 log cycles; and (3) a dose of 3 X 105 rad reduced the number by 3.7 log cycles. Values received for the effect of different levels of radurization are in fairly close agreement with those reported by Sinnhuber and Lee and cited by Ronsivalli [12]. The irradiation is on the whole more bactericidal than blanching and does not appear to be dependent on initial number of micro-organisms present. Only slight or insignificant variation in killing effect was found at each dose level, which is to be expected, as penetration of the lethal agent (gamma photons) is more effective than with heat. When blanching and irradiation treatment was combined, the total reduction of initial organisms was about 4-6 log cycles which resulted in all instances in bacterial numbers which were less than 100 per gram of material.

3.3. Results of storage tests at 0° - 1°C and 5° - 6°C on the effect of various treatments on the total bacterial count (TBC), sensory evaluation (T-tests) and volatile acid number (VAN)

3.3.1. The effect of irradiation only at different dose levels

Tables I, II and VIII and Figs 1 and 2 show on a comparative basis thé results-of different levels of irradiation on the three storage life criteria used at two storage temperatures. It is obvious that the storage

27 temperatures used (0° - 1° and 5° - 6°C) greatly influence the product1 s storage life. It may also be seen that irradiation prolongs the storage life at both storage temperatures. Increased radiation dose results in prolonged storage life. As can be expected, the killing rate of the highest dose level used, 3X 105 rad, is the greatest. The data show a significant difference between 1 X 105 rad and 3X 105 rad irradiation, whereas results of the 2X 105 rad irradiation lie in between. Thus at both storage temperatures it was found that an irradiation dose of 1 X 105 rad showed a two- to three-fold increase and a dose of 3X 105 rad gave up to a four-fold increase in storage life, compared with the untreated raw material. As stated above (Section 3.1.1) all the irradiated samples had blackened to various degrees long before the termination of the experiments which was decided by the three criteria where appearance was not included.

3.3.2. The effect of blanching only

Blanching for 1 and 2 min, respectively, did prevent blackening of the lobster tails except in a few individual cans which had been blanched for 1 min and given a dose of 3 X 105 rad. The product storage life resulting from a blanching time of 1 and 2 min was noticeably shorter at the higher storage temperature (Tables III - VI, VIII and Figs 3 - 6). Blanching for 1 min showed marginal storage life extension at both temperatures whereas 2-min blanching showed at least a two-fold increase. Samples stored for a few weeks beyond the end of the experiment showed no signs of blackening when the product had been blanched for 2 min. The reason for the longer storage life of the product blanched for 2 min compared with 1 min may be attributed to more effective killing of the microflora at the longer blanching time (see Section 3.2).

3.3.3. The combined effect of blanching and irradiation at different dose levels

Combination of the two treatments, blanching and irradiation, in all instances showed a longer storage life than individual treatments at comparative levels (see Tables and Figures). The killing rate of the combined treatment was greater than that of the individual treatments (see Section 3.2). The number of surviving micro-organisms was in all instances less than IX 102. Nevertheless, there was a significant difference in the storage pattern and storage life between individual groups of combined treatments when blanching time was constant but doses varied at the same storage temperatures. The storage life at the higher storage temperature was found to be about one half of that found at the lower storage temperature. One-min blanching time gave shorter storage life of the product than blanching for 2 min at corresponding levels of irradiation. According to the results obtained in the experiments with irradiation only (see Section 3.3.1) it was found that irradiation added to the storage life. The difference between the dose levels was, however, less obvious in the combined treatments than when irradiation alone was applied.

28 Storage life at 0° - 1°C storage temperature, when the product was blanched for 1 min and irradiated, was found to show a three- to five- fold increase compared with the untreated reference samples. Blanching for 2 min in conjunction with irradiation gave up to a six-or seven-fold increase in storage life. A similar relationship in the extension of storage life appears to hold for storage at 5° - 6°C.

4. DISCUSSION

It may be seen from the data that the quality of the raw material differed considerably from one batch to another. It would have been desirable to use raw material of similar quality and freshness. As, however, the raw material was obtained from commercial freezing plants after accepted methods of preparation, i. e. washing, degutting and grading, it was felt that the same methods of preparation would apply to a product intended for radiation preservation. Experiments, in which the same treatments were repeated but with different batches of raw material do not appear to indicate contradictory values for storage life. It was therefore necessary, because of the complexity of processing and storage factors under investigation, to pool the data from different dates of experiments and product batches. Though not ideal, this seemed to provide a valid basis for comparison.

4. 1. The radiation dose

The application of ionizing radiation at pasteurization dose levels to extend the storage life at refrigerated temperature must aim at a radiation dose that appears likely to make the product acceptable to the consumer and results in a relatively great extension of storage life. The product quality should be high and uniform. The literature [7, 13, 14] reveals that pasteurization radiation doses most commonly used for seafoods range from 0 . 5 X 105 - 3 X 105 rad. Preliminary experiments indicated that the dose of 3 X 105 rad was the upper threshold dose for lobster tails. Above this dose level typical radiation off-odours and off-flavours were pronounced. The selection of the lower limit dose of 1 X 105 rad as being the minimum effective dose for seafoods at refrigerating temperature was made on the basis of a literature survey [13, 14]. As stated above, radiation doses used in this study did not prevent the enzymatic melanosis of the product, whereas blanching for one or two minutes did.

4. 2. Sensory evaluation (T-test)

As indicated in Section 2. 2. 1, the sensory score resulted from the odour and flavour evaluation carried out by the taste-testing panel. The panel consisted of five-to-seven people who may be considered as expert tasters of seafoods. Since they are accustomed to fresh seafoods, their sensory evaluation may be regarded as stricter than that of the normal consumer. In the beginning a light aversion was expressed by the panel

29 members to the irradiation odour and flavour of the product. In a short time, the panel members became accustomed to this. They could easily pick out an irradiated product which had received the highest dose. On these grounds samples which had received 3 X 10s rad generally scored slightly lower than other samples. As can be expected there were some variations in scoring between the individual panel members and some day-to-day variation of the average score on the same sample. The size of the individual lobster tails may account for some of the daily and day-to-day variation in scoring. How- ever, a close agreement in the scoring of the product towards the end of storage life (borderline value) was evident. The borderline for the sensory evaluation was as a rule reached at a later date than the microbial limit of 1 X 106 bacteria per gram. Taste-testing of samples stored at 5°-6°C was discontinued as soon as there was any sign of putrefaction in which case odour evaluation only was done. This was exercised as a precautionary measure to protect panel members from possible food poisoning.

4. 3. Volatile acid number (VAN)

In a paper by Spinelli [8] the author discusses critically the use of chemical test methods for quality assessment of irradiated seafoods. Of the great number of chemical tests proposed as quality indices for fresh and stored seafoods the author suggests that only two or three tests are of some value for irradiated seafoods. These tests are trimethylamine (TMA), total volatile basis (TVB) and volatile acid number (VAN) determinations. The VAN test was the method which the author believes is the most likely to give indicative results. Because of lack of personnel a choice had to be made, limited to one test if possible. The VAN test was chosen, as it is easy to carry out and does not require elaborate laboratory equipment. During the course of this study it was seen that this test is, to say the least, of limited value as a quality index for irradiated lobster tails. This applies also to the product blanched for 2 min. On the other hand, the test appears to be applicable for a product blanched for 1 min or less. For the untreated product the results showed that the VAN test is indicative of the spoilage pattern. Tests made on the raw material could also distinguish between different states of freshness. Table VIII shows the VAN value at the upper time limit of product acceptability according to the sensory evaluation. In general, VAN test results given in this table were determined on the day the experiment was considered finished. When surplus samples were available the VAN test was continued far beyond the borderline point. As an example, it may be cited that samples that had been blanched for 2 min and then irradiated were tested up to 80 days, which was about twice the storage life of the product. This added storage time revealed no increase in VAN value. On a few occasions it was observed that samples, particularly those that received a dose of 1 X 05 rad and were not blanched, showed an increase in VAN value when stored beyond the limits of acceptability.

30 4. 4. Bacteriological aspect of the study

The culture medium used in this study was that proposed by Liston [10] with some modifications. Other workers [13] have used a similar culture medium (Eugonagar) which contains reducing substances. For a part of this study, a comparison of the total bacterial counts was made by using Plate Count Agar (Difco). Results of this comparison are not included in this report. The temperature of incubation used for determining total bacterial counts was 22°C (room temperature). Bacterial counts made after incubation at this temperature presumably detect the total psychrophilic bacterial flora. Bacterial counts made on lobster samples which had received the combined treatment of blanching and irradiation were unable to detect the mesophilic flora when plates were incubated at 3 7°C and showed no growth. However, if samples were removed from storage of 0°-l°C and left at room temperature for 24 h or longer and then tested bacteriologically, very high counts were found after incubation at both 22°C and 37°C. The seafood product — lobster tails — under study comes from an ocean environment where the temperature seldom exceeds 10°C. The microbial flora is therefore predominantly psychrophilic in nature. It is the chief cause of spoilage of the product, even though preservation methods followed by low storage temperature are applied. Pathogenic organisms are mostly introduced to seafoods by contamination from workers during handling and processing, the exception being certain Clostridia species, such as Cl. botulinum type E which has been found in freshly caught fish. CI. botulinum type E is able to produce toxin at a low storage temperature [14, 15]. With these facts in mind the storage temperature of choice is 0°-l°C. The temperature of 5°-6°C simulated conditions believed to prevail in both commercial and domestic refrigerators. Colby and Shewan [16], Ronsivalli [13] and Dassow [17] have discussed the qualitative and quantitative microbial aspects of pre- and post- irradiation of fish and fish products. In this study no attempt was made to identify the bacterial species found before and after irradiation or blanching and irradiation. This should be included in further work on radurization of lobster or lobster tails. The bacteriological research in this study is therefore limited in scope, and is used along with other criteria for comparison and assessment of storage life of the lobster product under simulated commercial conditions. It can be seen from the tables and Fig. 7 that the initial number of bacteria varied considerably in the raw material counted after 72-h incubation at 22°C. In some of the experiments the bacterial count exceeded 1 X 106per gram. This could not be helped as the raw material was received from commercial sources at a stage when it was ready for packaging and freezing. As will be referred to below, some information was obtained on bactericidal efficacy of blanching using raw material of different initial bacterial counts. The limits of acceptability as far as bacterial counts were concerned was set at 1 X 106 per gram. At this limit untreated lobster tails are on the whole judged as being of poor eating quality. As the experiments progressed it became evident that the limit of .1 X 106 bacteria per gram

31 of treated product corresponded poorly with sensory evaluation, which in most instances indicated longer storage life than the bacterial count of 1 X 106 indicated. Even though not determined it can be stated that most frequently the lobster had reached counts of 1 X 107 or higher when it had reached a borderline of acceptability as determined by sensory evaluation. Ronsivalli [13] and Dassow [17] have observed similar results in their study of irradiation of fish and fish products. In Section 3. 2 the results of bactericidal efficacy in this study are given (see Fig. 7), i. e. the bactericidal efficacy of pasteurization doses of irradiation and heat treatment (blanching) either as a single or as a combined determinant. The two bactericidal processes as applied here differ in nature. The radiation process applied here is pasteurization (radurization) of the product. This treatment aims at reducing and controlling spoilage bacteria but control of pathogenes may or may not be accomplished by these doses. The heat treatment (blanching) is applied to prevent melanosis. The bactericidal effect of blanching is inherent in but incidental to the process. As mentioned in Section 3. 2, blanching for two minutes showed variations in bactericidal effects, and it was suggested that the effects depended on the initial number of micro-organisms, this being explained on the basis of the localization of the micro-organisms in the lobster tissue. It was to be expected that the killing would be most effective on the surface of the lobster tail pieces. The heat is less penetrating and damaging than the gamma photons. Under Section 3. 3 results are given of storage tests at 0°-l°C and 5°-6°C on the effect of various treatments (see Tables I-VIII and Figs 1-6). From the combined data the relative storage life was determined (see Table VIII). The results showed that storage life at 5°-6°C is about half of that at 0°-l°C. This is due to differences in duration of the lag phase. After storage at 5°-6°C the lag phase was on the whole short apart from the fact that the combined treatment of blanching for 2 min and a dose of 3 X 105 rad showed some extension of the lag phase. At the lower storage temperature (0°-l°C) the lag phase was noticeably long (20 days) when the product was blanched for 2 min and received a dose of 3 X 10s rad. The lag phase observed with 1-min blanching and irradiation was shorter, and still shorter when irradiation alone was used. From the microbiological point of view combined treatment of 2-min blanching and dose levels of 2 X 105- 3 X 105 rad appear to be the most promising. The effect of the packaging material on the microbial pattern was not dealt with in the report. Ronsivalli [12] has raised some very basic and interesting points on the matter. Some of Ronsivalli's questions, concern this study, since hermetically sealed containers were used for packaging. How and to what extent this type of container affected the results on bacterial counts is not known.

4. 5. Comparison of factors influencing storage life

Results of storage studies show that of the two storage temperatures used.the one at 0°-l°C gave twice as long an extension of storage life as that at 5°-6°C. From the data a maximum storage life of the product for about 2 to 3 weeks, after combined treatment, may be expected at the higher storage temperature (5°-6°C). The product kept at this temperature

32 (5°-6°C) should not be intended for a long-distance shipment. At this temperature the product receiving irradiation only had a storage life of one week, whereas the product storage life was extended to 2 to 3 weeks when irradiation and blanching for 2 min was used. Untreated reference material as generally received in this test and stored at 5°-6°C had a storage life of 1 to 2 days. Freshly caught untreated lobster is generally unacceptable after 5 to 6 days when kept on ice. Irradiated lobster samples had a storage life of up to 3 weeks at a storage temperature of 0°-l°C. By blanching for 1 min and then irradiating the lobster tails an extension up to a month was obtained. The best storage life was obtained when the lobster tails were blanched for 2 min and then irradiated; this treatment resulted in storage life of about 6 weeks. This is a two-fold increase in storage life over both irradiation treatment only and blanching for 2 min only.

4. 6. Possible use of irradiation for processing and preservation of lobster

The lobster fisheries in Iceland have been described in the introduction. At present only a few fish freezing plants, at a relatively short distance from each other, are engaged in processing lobster. This area is also the centre of the whitefish freezing industry, providing an abundant supply of fish as raw material for various processing methods. As a future site for a commercial irradiation facility this locality seems to be the logical one. The lobster fishery is seasonal. The whitefish fishery has a peak season at another time of the year but functions all the year round. This constitutes the basis for a commercial irradiation plant that would operate throughout the year. The operation of such a facility would necessitate considerable modifications in handling of catch, pre-irradiation treatment and packaging of the product. It should be realized, however, that much work needs to be done before its inception. A yearly production capacity of at least 5000-6000 metric tons might be expected. Of this, the lobster tails would constitute about 1000 metric tons, and would have to be processed within three months, whereas the whitefish and other seafood products would make up the raw material for the remainder of the year. An irradiation facility of this size has been proposed in the literature [18, 19] as the minimum size of plant believed to be operational and economically comparable with other food processing establishments. The major conclusion reached from this study is that the storage life of lobster tails kept at 0°-l°C is substantially extended by the combined treatment of blanching and irradiation. Iced, raw lobster is for obvious reasons not exported from Iceland unless frozen. By using the suggested combined treatment a fresh lobster product may be foreseen as a commodity on the foreign market, now dominated by the frozen product. It is very difficult to evaluate, when making a comparison with the existing processing methods, what economic implications such a fresh product would have for the lobster industry in Iceland. Further speculation in this regard is futile until consumer acceptance has been surveyed, and the food-laws of prospective market countries have approved radurized seafood products [20].

33 5. SUMMARY AND CONCLUSIONS

From the experimental data the following pertinent points may be made. The validity of the results obtained by the test criteria used in this study indicate that sensory evaluation (5-point T-test) and total bacterial count at 22°C are in fairly good agreement and can be used for determining storage life. Volatile acid number determinations on the irradiated product (2 X 105 - 3 X 105 rad) and the product blanched and irradiated gave unreliable results. This test, however, has its value for the untreated product and possibly for the product receiving irradiation doses lower than 2 X 10s rad. Results obtained by using irradiation only gave up to a three-fold increase in storage life according to sensory and bacterial determinations. If judged by appearance storage life would be much less, as all dose levels of radiation did not prevent melanosis and the highest dose seemed to increase it. By blanching the product for 2 min melanosis was completely inhibited. The blanching treatment also reduced the number of bacteria in the raw material considerably. It must be realized that the physical appearance of the lobster meat is somewhat changed by blanching and takes on the appearance of a cooked product at the cut surface. The colour of the shell is lightened also. Consequently, the most promising results found in the study were those when the product was blanched for 2 min and then irradiated, preferably 5 at a dose level of 2 X 105 - 3 X 10 rad. Storage life of such a product was found to be about 6 to 7 weeks. Storage temperature of 0°-l°C is recommended for such a product. The borderline of acceptability for the total bacterial count was in this study set at 1 X 106 bacteria per gram which appears to be too low when compared with taste acceptability. The prolonged storage life of the highly priced lobster tails which results from applying heat and radiation pasteurization preservation could lead to new markets for this product. The ramification of these possibilities are discussed in the body of the report. The authors suggest that future research on radiation preservation of Norway lobster should include the following:

(1) The effect of raw material quality on the storage life. (2) Identification be undertaken of bacterial species associated with the pre- and post-irradiated product. (3) Study be undertaken on the feasability of blanching whole lobster at sea and processing and irradiating on shore. (4) Possibilities of utilizing ionizing radiation in the Icelandic fish industry including installation, handling, production and packaging costs. (5) Consumer acceptance and market prospects for blanched, irradiated lobster product should be examined, including commercial export trials to foreign markets.

34 PART II RADURIZATION OF DEEP-SEA SHRIMP

6. INTRODUCTION

The second phase of the irradiation studies on sea foods deals with radiation pasteurization of deep-sea shrimp (deep-sea prawn, Pandalus borealis). This product is widely distributed in nature and of world-wide commercial interest. The deep-sea shrimp caught off the Icelandic coast is a small species, the meat portion amounting to 1-2 g, and is a highly priced product which resembles lobster in many respects such as price, availability and spoilage pattern. The deep-sea shrimp fisheries started about 30 years ago but have attained commercial importance during the past few years. The average annual catch is about 2000 metric tons. The main catching grounds are off the northwest coast of Iceland but it is also found in other coastal areas [21]. The deep-sea shrimp season is principally from October until the end of April. The shrimp is not cooked on-board but kept iced until landed. It is stored on ice until cooked and peeled. There are two methods of peeling, by hand and by machine. Hand peeling is done after cooking the shrimp in water; the cooking is carried out shortly after landing, usually in a brine of 5% salt. The brine may also contain other preservatives, e. g. sodium benzoate, for use with this product which is sold to the Scandinavian market. Machine peeling is carried out after at least 24-h storage on ice and before cooking in steam. This product is mostly deep-frozen but some is canned. The frozen product without the preservatives is sold mainly to the United Kingdom and Continental Europe. The object of this study was to evaluate the possible application of the radiation preservation of deep-sea shrimp in comparison with frozen shrimp. To accomplish this, various factors associated with pre-irradiation treatment of shrimp, irradiation of shrimp and storage study of the product were studied. This report may be considered to be a sequel to Part I, to which direct reference will be made in relation to methods of preparation, methods of analysis, etc. Deep-sea shrimp investigations carried out by Danish workers have been most helpful in this study [22, 23]. Literature pertaining to general aspects of the irradiation of shellfish has been cited in Part I.

7. MATERIALS AND METHODS

7. 1. Materials

7. 1. 1. Raw material, handling and preparation

The raw material used was obtained chiefly from shrimp processing plants shortly after landing and shipped iced by air to the laboratory.

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37 The shrimp was on the average about 24-h old when experiments were started. Raw material quality was found to vary somewhat which undoubtedly affected the overall results of the storage studies. After the shrimp was received at the laboratory it was in most instances blanched for different lengths of time, hand-peeled, packed in polyethylene bags and in some cases irradiated, before storage at 0°-l°C. The study was organized into five groups of experiments, which un- fortunately do not always represent the same raw material all through the group. Because of the complexity of the processing data sought in this study, separate trials had to be done. The grouping is outlined in Table IX and was as follows.

(1) The first group dealt with a storage study on the effect of various treatments before peeling and irradiation. This involved blanching for 2 and 5 min, low-dose pre-irradiation (0. 6 X 105 rad) and storage at 0°-l°C of the unpeeled shrimp. The effect of these pretreatments was compared with an untreated control sample (Table X, Figs 8-10).

FIG. 8. Comparison of the effect of different pre-irradiation treatments on the storage life of unpeeled shrimp as determined by the total bacterial count (TBC). Storage temperature 0°-l°C.

38 (2) The second group involved study of the effect of blanching for 2 and 5 min and subsequent peeling and storage at 0°-l°C (Table XI, Figs 11- 13). (3) The third group of experiments involved a comparison of the effect of combined treatments, i. e. double-dose irradiation (pre-irradiated with 0. 6 X 105 rad in the shell and 2 X 105 rad after peeling), blanching for 2 min and irradiation subsequently with dose levels of 1 X 105 and 2 X 105 rad, and finally blanching for 5 min and subsequent irradiation at a dose level of

1 X 105 rad (Table XII, Figs 14-16).

15 20 days

FIG. 9. Comparison of the effect of different pre-irradiation treatments on the storage life of unpeeled shrimp as determined by the sensory evaluation score (T-test). Storage temperature 0°-l°C.

39 (4) In the fourth group unpeeled shrimp, blanched for 2 min, was stored at 0°-l°C for 2, 5 and 7 days, then peeled and irradiated at a dose level of 1 X 105 rad and storage continued. The effect of the pre-irradiation storage on the total storage life was determined (Table XIII, Figs 17-19). (5) The fifth group of experiments consisted of irradiation of commercially pre-cooked, brine-dipped, peeled shrimp.

7.1.2. Blanching

The blanching operation was performed as described in Part I, Section 2. 1. 4, by immersing the shrimp in boiling water.

shrimp as determined by trimethylamine (TMA). Storage temperature 0°-l°C.

40 TABLE XI. COMPARISON OF THE EFFECT OF 2- AND 5-MIN BLANCHING ON STORAGE LIFE OF PEELED SHRIMP AS JUDGED BY THE TOTAL BACTERIAL COUNT (TBC), SENSORY EVALUATION SCORE (T-TEST) AND TRIMETHYLAMINE DETERMINATION (TMA); STORAGE TEMPERATURE 0°-l°C

Days Blanching Blanching of (2 min) (5 min) storage TBC T-test TMA TBC T-test TMA

0 7.1 X 103 3.1 0.3 2 X 101 4.0 0.5

3 2 2.2 X 10 - -

4 1. 3 X 102 3.2 0.4 1.3 X 103 3.1 0.7

6 1. 8 X 103 2.7 0.8

8 3.2 X 103 2.9 0.2

9 3. 7 X 104 3. 3 0.5

11 1. 9 x 104 3.2 0.8

6 12 1. 7 X 10 2.1 (2. 3) a

15 4.1 x 107 1.0 0.7

16 6. 6 X 107 2.0 0.6

20 1.0 0.4

a Value not used for compiling Fig. 13.

7. 1. 3. Packaging

The packaging material used was polyethylene plastic bags of 0.07 mm thickness. About 80-100 g of the peeled shrimp were packed in plastic bags which were then heat-sealed without vacuum. The unpeeled shrimp, about 250-300 g, was also put into the same type of pouches, sealed, wrapped with aluminium foil and put into a second outer bag which was also sealed. This was necessary to prevent loss of liquid as the antennae tended to pierce holes in the inner pouch.

7. 1. 4. Storage

All samples were stored at 0°-l°C.

41 \

Í<3 7 ID

Ш6 ГЭ Z о о

15 20 days 20 days FIG.11. Comparison of the effect of blanching for FIG. 12. Comparison of the effect of blanching for 2 and 5 minutes on the storage life of peeled 2 and 5 minutes on the storage life of peeled shrimp as determined by the total bacterial count shrimp as determined by the sensory evaluation (TBC). Storage temperature 0°-l°C. score (T-test). Storage temperature 0°-l°C.

7. 1. 5. Irradiation

Dosimetric determinations were carried out by inserting 10 vials containing Fe/Cu solution in between pouches of shrimp. When the shrimp pouches did not take up all the space in the irradiator container, the pouches were placed in the centre of the container and the empty space filled with water bags. The average dose rate was found to be about 420 rad/sec. The dose levels of irradiation used were 0 . 6 X 105, 1 X 105 , 2 X 105 and 3 X 105rad.

42 7. 2. Methods

7. 2. 1. Bacteriological determinations (TBC)

Bacteriological testing of the shrimp was carried out in a manner similar to that described in Part I, Section 2. 2. 3, using the same culture medium. When peeled shrimp was tested only 22 g instead of 44 g were used and appropriate serial dilutions made. The bactericidal efficacy of the various pretreatments and dose levels of irradiation were also determined. Incubation temperature was 22°C.

7. 2. 2. Sensory evaluation (T-test)

Sensory evaluation was carried out in a .similar manner to that described in Part I, Section 2.2. 1, using the 5-point scale and the same group of panel members. The sensory evaluation differed only in the method of preparing samples for tasting: the shrimp samples were steamed in closed glass containers instead of boiling in water.

7. 2. 3. Trimethylamine determination (TMA)

In Part I it was stated that the volatile acid number determination (VAN) was of limited value for assessing the quality of blanched, irradiated

FIG. 13. Comparison of the effect of blanching for 2 and 5 minutes on the storage life of peeled shrimp as determined by trimethylamine (TMA). Storage temperature 0°-l°C.

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44 FIG. 14. Comparison of the effect of various combined treatments of blanching and irradiation on the total bacterial count (TBC). Storage temperature 0°-l°C.

lobster tails. Because of the apparent resemblance of the two seafood products it was decided to attempt to use trimethylamine determination as a means of assessing the chemical quality index. The method used was that of Dyer [24] with minor adaptations. Twenty- five grams of minced shrimp were mixed with 50 ml of 7.5 per cent trichloroacetic acid and filtered. To a 1 ml aliquot of the filtrate was added 4 ml of 5 per cent trichloroacetic acid, 1 ml of formaldehyde solution, 10 ml of toluene (specially purified) and 3 ml of saturated K2CO3. The mixture was shaken 40 times and left to stand for 10 min. A 5 ml portion of the toluene layer was dried with Na^SQj and 5 ml of 0.02 per cent picric acid in toluene were then added. The yellow colour intensity was determined in a Beckman DU spectrophotometer at a wave- length of 410 щи against a blank. Results are expressed as mg N TMA/100 g fish.

45 FIG. 15. Comparison of the effect of various combined treatments of blanching and irradiation on the sensory evaluation score (T-test). Storage temperature 0°-l°C.

8. RESULTS

8. 1. Bacteriological determination (TBC)

8. 1. 1. The effect of processing treatments on the number of surviving bacteria (bactericidal efficacy)

In this part of the study the number of surviving bacteria after various experimental treatments before final storage was determined in a manner similar to that described in Part I, Section 3. 2.

46 FIG. 16. Comparison of the effect of various combined treatments of blanching and irradiation on trimethylamine (TMA).

The bactericidal efficacy of pasteurization doses of irradiation and heat treatment (blanching) was determined either as a single factor or more often as a combined factor. The results are shown In Table XIV. The log number of initial bacteria present in the raw material per gram ranged from 6.22 to 4.97, averaging 5.65. The average reduction in bacterial numbers when blanching treatment alone was used was as follows:

(i) blanching for 2 min reduced bacterial numbers on the average by 3.89 log cycles; (ii) blanching for 5 min reduced bacterial numbers on the average by 4.03 log cycles (two trials) (see Table XIV, groups A,B,C,D and E).

Data on reduction of bacterial numbers when irradiation only was applied is scant in this study as only one trial was made using 0.6 X 10s rad followed by 2 X 105 rad 2 days later after the shrimp had been peeled.

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48 FIG. 17. Comparison of the effect of storing unpeeled shrimp, blanched for 2 min, at 0°-l°C for 2, 5 and 7 days before peeling and irradiation at 1 x l№ rad on the total bacterial count (TBC). Storage temperature 0°-l°C.

The reduction of bacterial numbers by a dose of 0.6 X 105 ra(j was 0.59 log cycles. An additional reduction of 1.12 log cycles was found after 2 days when the peeled shrimp was given a second dose of 2 X 105 rad (see TableXIV, group A). When blanching and irradiation treatment were combined the reduction in number of bacteria from the initial count was:

Blanched for 2 min, peeled and irradiated at a dose level of 1 X 105 rad the same day, the reduction was 4.92 and 2.83 log cycles in each of two experiments (TableXIV, groups В and D); at a dose level of 2 X 105 rad it was 3.10 log cyles (TableXIV, group D) in one trial.

It should be noted in respect to the last two log values (TableXIV, group D) that the shrimp became recontaminated after blanching when peeled. This recontamination factor was not always determined (see TableXIV, group B).

49 FIG. 18. Comparison of the effect of storing unpeeled shrimp, blanched for 2 min, at 0°-l°C for 2, 5 and 7 days before peeling and irradiation at 1 X 105 rad on the sensory evaluation score (T-test). Storage temperature 0°-l°C.

The reduction of bacterial numbers when the product was blanched for 5 min and then irradiated by 1 X 105 rad is uncertain as the irradiation was done after 2 days storage of the shrimp in shell (Table XIV, group E). In Table XIV, group C, is shown the effect of keeping blanched shrimp in shell for 2, 5 and 7 days respectively, after which time each batch was peeled and then irradiated at a dose level of 1 X 105 rad before the final .storage study was made. The bactericidal efficacy of blanching for 2 min before storing the shrimp in shell has been reported above. However, it should be pointed

50 FIG. 19. Comparison of the effect of storing unpeeled shrimp, blanched for 2 min, at O'-l'C for 2, 5 and 7 days before peeling and irradiation at 1 x 10s rad on trimethylamine (TMA). Storage temperature O'-l'C.

out that the actual overall bactericidal efficacy of the combined treatment of blanching followed by irradiation is very difficult to assess because of an increase in bacterial numbers (recontamination) during pre-irradiation storage and because of contamination during peeling. It may be seen, however, that the number of surviving bacteria after storage of 2, 5 and 1 days followed by irradiation was in all three instances less than 100 per gram before determination of storage life. Table XIV, groupF, shows the bacterial changes of commercially pre-cooked shrimp in shell during 3days' storage and subsequent irradiation at a dose level of 2 X 105 rad.

51 TABLE XIV. LOG NUMBER OF SURVIVING BACTERIA AFTER VARIOUS EXPERIMENTAL TREATMENTS OF SHRIMP BEFORE FINAL STORAGE AT 0°-l°C

Log cycle Log number variation survivors Increase

Group A

(1) Unirradiated (0 rad) 6.11 (2) Blanched 2 min in shell 1.48 4. 63 (3) Irradiated 0. 6 x 10s rad in shell 5.52 0.59 (4) Irradiated 0.6 X 105 rad in shell kept 4 d and Irradiated 2 X 105 rad 4.40 1.12 (5) Blanched 2 min, kept 4 d and irradiated 2 x 105 rad 1.60 0.48

Group В

(1) Unirradiated (0 rad) 6.22 (2) Blanched 2 min in shell 3.21 3.00 (3) Blanched 2 min in shell, peeled 1.30 1.90 and Irradiated 1 x 105 rad

Group С

(1) Unirradiated (0 rad) 5. 60 (2) Blanched 2 min in shell 1.48 4.12 (3) Blanched 2 min in shell, kept 2 d 1. 78 0. 30 (4) Blanched 2 min in shell, kept 2 d, 0.18 1.30 peeled and irradiated 1 x 105 rad 0.48 (5) Blanched 2 min In shell, kept 5 d 2.70 1.22 (6) Blanched 2 min in shell, kept 5 d, 4.43 2.95 peeled (7) Blanched 2 min in shell, kept 5 d 1.30 0.18 peeled and irradiated 1 x 10s rad (8) Blanched 2 min in shell, kept 7 d 4.10 2.62 (9) Blanched 2 min in shell, kept 7 d, 3.10 1.62 peeled (10) Blanched 2 min in shell, kept 7 d, 1.48 peeled and irradiated 1 x Ю5 rad

Group D

(1) Unirradiated (0 rad) 5. 39 (2) Blanched 2 min in shell 1. 60 3. 79 (3) Blanched 5 min In shell 1. 00 4. 39 (4) Blanched 2 min in shell, peeled 3. 85 (1. 54) 2.25 (5) Blanched 2 min In shell, peeled 0.96 and irradiated 1 x 105 rad 2. 56 1.29 (6) Blanched 2 min in shell, peeled 0. 68 and Irradiated 2 X 105 rad 2.28 1.57

52 TABLE XIV (cont. )

Group E

(1) Unirradiated (0 rad) 4. 97 (2) Blanched 5 min in shell 1. 30 3. 67 (3) Blanched 5 min in shell, kept 2 d 1.63 2.14 and peeled 3.34 Г 0.30 (4) Blanched 5 min in shell, kept 2 d, 3. 97 peeled and irradiated 1 x 105 rad 1. 00 \ 2.34

Group F

(1) Commercially pre-cooked in shell 3. 68 (2) Commercially pre-cooked in shell, 3.34 peeled (3) Commercially pre-cooked in shell, ' 1.00 2.68 peeled and irradiated 2 x 10s rad (4) Commercially pre-cooked in shell, 4. 38 0. 70 kept 3 d (5) Commercially pre-cooked in shell, kept 3 d, peeled and 1.48 2.90 2.20 irradiated 2 x 105 rad

8. 2. The effect of various processing treatments and irradiation on storage life

8. 2. 1. The effect of various treatments before peeling and irradiation on storage at 0°-l°C

Results of this experimental group may be found in Table X and Figs 8-10. The purpose of this experiment was to evaluate which pre- irradiation treatment would be thè most suitable if radiation preservation were to be used in the Icelandic shrimp industry. The untreated control samples, held at 0°-l°C, were found to have a storage life of 5-6 days. Similar results are usually obtained in industrial practice, when shrimp is- kept on ice. The first indication of spoilage was evidenced by green to black discolouration. Low-dose initial irradiation of 0.6 X 105 rad on unpeeled shrimp was found to be ineffective in preventing discolouration and delayed spoilage insignificantly. The effect of double-dose irradiation, i. e. initial low dose and high dose repeated several days later, will be discussed in Section 8. 2. 3. It is customary in the shrimp industry to use blanching or pre-cooking to facilitate peeling and prevent discolouration. In Part I blanching for 2 min in boiling water gave good results on lobster tails. It was therefore decided to try the same approach here. Blanching unpeeled shrimp for 2 min gave a storage life of about 10 days. Discolouration of the head portion became noticeable after about 7 days storage, but it was not evident in the meat portion after peeling at that time. Increasing the blanching time to 5 min whereby.the shrimp was placed in boiling water but the cooking water did not reach boiling temperature again, was then tried in order to prevent this discolouration. The discolouration of the

53 TABLE XV. STORAGE LIFE OF SHRIMP AFTER VARIOUS TREATMENTS, AS JUDGED BY TOTAL BACTERIAL COUNT (TBC), SENSORY EVALUATION SCORE (T-TEST) AND TRIMETHYLAMINE VALUE (TMA), AT END OF EXPERIMENT; STORAGE TEMPERATURE 0°-l°C

TBC2 T-test TMA at 1 x 106 s2 end of Sample treatment before storage after after experiment days a days

Group 1: stored unpeeled

Untreated control 7 5 5 s Pre-irradiated with 0. 6 X 10 rad - 6 1 Blanched 2 min 10(11) 11 8 Blanched 5 min 12 (14) 17 4

Group 2: stored peeled

Blanched for 2 min 13 (14) 14 0.5 Blanched for 5 min 12 (14) 16 0.5

Group 3: stored peeled Irradiated with 0. 6 x 10s + 2 x 105 rad 18 (19) 5 1 Blanched 2 min + 1 x 105 rad 21 (25) 41 4 Blanched 2 min + 2 x 105 rad 34 ( 39) 42 1 Blanched 5 min + 1 x 105 rad 41(45) 46 1

Group 4: stored blanched, unpeeled until irradiation

Blanched 2 min + 1 x 105 rad at 0 days 21 (25) 41 4 Blanched 2 min + 1 x 10s rad after 2 d 31 (34) 42 0.5 Blanched 2 min + 1 x 105 rad after 5 d 31 (40) 40 10 Blanched 2 min + 1 x io5 rad after 7 d 26 (32) 35 1

a Values in parentheses are TBC when borderline of acceptability is raised to 1 X 107. head portion was, however, noticeable after about the same length of storage (8 days) so increasing the blanching time to 5 min did not appear to be determinative in this respect. The storage life of the shrimp blanched for 5 min could, however, be extended to 12 days according to the TBC test and up to 17 days according to the T-test and TMA values, if physical appearance (discolouration) is disregarded (see Table XV).

8. 2. 2. The effect of blanching for 2 and 5 min and subsequent peeling on storage at 0°-l°C

The purpose of these experiments was to test how long blanched and peeled shrimp could be kept at 0°-l°C. The results of these experiments are to be found in Table XI and Figs 11-13. Blanching for 2 min resulted in storage life of 12-13 days for shrimp peeled immediately after blanching. When the blanching time was 5 min the storage life was similar, 12-15 days.

54 8. 2. 3. The effect of combined treatments on storage life of peeled shrimp at 0°-l°C

Results of these experiments are to be found in .Table XII and Figs 14-16. The purpose of the experiments was to find the optimal combination of pre-treatments and irradiation for extension of storage life. As mentioned above (see Section 8. 2. 1), blanching of the shrimp was found to be necessary to prevent discolouration. Nevertheless it was found worthwhile to include here one trial where the product, pre-irradiated with 0.6 X 105 rad, was given a second dose of 2 X 105 rad after peeling. As may be seen in Table XII, this sample was objectionable to the panel members after 8 days of storage, long before the total bacterial count and TMA value indicated spoilage. When the dose level was increased to 3 X lOSrad, radiation off-odour and off-flavour were so pronounced that it was felt it would be out of the question to use this dose level commercially. Textural changes were also very noticeable and objectionable as the meat became soft and mushy. At a dose level of 2 X 10s rad some of these organoleptic changes could be detected but to a lesser degree. This resulted in lower organoleptic scoring than was found for 1 X 105 rad but the overall storage life is quite similar (about 40 days) for the product blanched for 2 min and irradiated at a dose level of 1 X 105and 2 X 105 rad. At a dose level of 1 X 10s rad only minor organoleptic changes occurred. This dose level was used on peeled shrimp previously blanched for 5 min. The combination of 5 min blanching and irradiation at a dose level of 1 X 105 rad resulted in storage life of about 45 days.

8.2.4. The effect on the total storage life of storing unpeeled, blanched shrimp at 0°-l°C for 2, 5 and 7 days before peeling and irradiation

Results of this group of experiments are to be found in Table XIII and Figs 17-19. The purpose of the experiment was to store unpeeled, blanched shrimp for 2, 5 and 7 days at 0°-l°C before peeling and irradiation, and to see how this pre-irradiation storage affected the post-irradiation storage life. Samples stored blanched and unpeeled for 2 and 5 days showed no significant difference in total storage life of about 40 days. These samples showed, however, significantly longer total storage life than the sample which was kept blanched and unpeeled for 7 days before irradiation. The sample peeled and irradiated immediately after blanching and used for comparison in the table and the figures behaved somewhat contrary to expectation, as its total storage life was according to T-test about the same as the samples stored for 2 and 5 days before irradiation. Post- irradiation storage life seems to be affected by pre-irradiation storage. The longer the pre-irradiation storage, the shorter is the post-irradiation storage life.

8. 2. 5. Irradiation of commercially pre-cooked, brine-dipped shrimp

A. batch of commercial shrimp, which had been pre-cooked for 8 min at unknown temperature in about 5% brine was obtained from a shrimp processing plant.

55 The batch was kept for 3 and 6 days at 0°-l°C before peeling and was the irradiated with 2 X 105 rad. The experiment was terminated shortly after its beginning because both the unirradiated and the irradiated samples showed yellow discolouration of the meat, and rancid taste and odour, all of which were intensified by irradiation. A salt content of 2% or more in the meat seems to induce these undesirable changes (unpublished data).

9. DISCUSSION

The raw material in this study differed somewhat from time to time, as may be expected as it was obtained commercially. This caused discrepancies in the results similar to those experienced in the lobster project (Part I). But as the entire project was dealing with the practical aspects of irradiation of seafood products it is logical to use the same raw material as industry uses. Exceptionally fresh and uniform raw material could have only been acquired by extra effort. The main shrimp fishing grounds in Iceland are off the northwest coast and shrimp processing plants are located in that part of the country. These^ are quite small and are at present 10 in number. As discussed in Part I, it was suggested that the food irradiation centre be located on the southwest coast of Iceland, where the main whitefish and lobster fisheries are located. This would mean transporting the shrimp from the west coast to the irradiation centre, a trip by.boat, which may last for about 12-24 h depending on loca- tion, etc. This again necessitates some pre-treatment such as blanching or pre-cooking which should be performed on board the boats. As may be seen from Table X and Figs 8-10, the results showed that low-dose pre-irradiation of shrimp in shell was ineffective in preventing discolouration and gave relatively short storage life. A pre-treatment method, which is already used in the shrimp industry, i.e. pre-cooking, is used mostly to facilitate peeling of shrimp, but also serves the purpose of retarding spoilage, and preventing discolouration before final processing, freezing or canning. In Part I similar heat treatment was found to be effective in prolonging the storage life of lobster tails, when used as a pre-treatment for irradiation. The same approach was used in this study except that blanching times were 2 and 5 min. Results showed that unpeeled shrimp that had been blanched for 2 min could be kept for about one week at 0°-l°C and for a longer period of time when blanching for 5 min was used. This pre-treatment, blanching, should suffice to preserve the unpeeled shrimp until it reaches the irradiation centre, where it would be peeled, packaged and irradiated. As stated in Sections 8. 2.1 and 8.2.3, irradiation alone on unpeeled or peeled shrimp does not prevent discolouration. The unappealing appearance and textural changes of the unblanched, irradiated shrimp would make this product unmarketable. Doses used on the blanched product were 1 X 105 and 2 X 10s rad, but a higher dose such as 3 X 105 rad was found unsuitable. Even the dose level of 2 X 105 rad was found objectionable by sonie panel members. This was due to both irradiation off-flavour and textural changes.

56 Comparison of the optimal combinations of treatments — blanching and irradiation - is presented in Table XII and Figs 14-16 and shows that blanching for 2 min and irradiation at a dose level of 1 X 105 or 2 X 105 rad gives a storage life of about 40 days according to the sensory evaluation (T-test). The difference between the dose levels of 1 X 10s and 2 X 105 rad could be detected by the bacterial counts and the TMA values. The 5-min blanching and 1 X 10® rad irradiation gives a somewhat longer storage life. Even though the longer blanching time of 5 min was not tested as intensively as the 2-min blanching it appears to merit further studies and might be suggested as the pre-treatment of choice. It should be pointed out that the longer blanching time might result in lowered product yield [22, p. 32] . It has been pointed out that the shrimp fisheries and processing plants are located at a considerable distance from the proposed site of the food irradiation centre. Consequently, it was felt logical to take this into account in the present study on shrimp. Therefore, storage of blanched, unpeeled shrimp at 0°-l°C for a period up to a week before peeling and irradiation was included in the study. Results show that blanched, unpeeled shrimp will keep up to 5 days before peeling and irradiation without affecting to any degree the quality of post-irradiation storage (see Section 8.2.4). In Figs 17-19 the results are expressed as total storage life including pre-irradiation storage. The post-irradiation storage life of the marke- table product which had been kept at 0°-l°C up to 5 days before irradiation was comparable with that which was blanched and irradiated the same day. It should be noted, however, that samples stored for 7 days before irradiation had considerably less post-irradiation storage life than those stored for 5 and 2 days. Since the distance involved is considerable it is essential to organize the transport of the blanched shrimp in such a way that the time elapsed between blanching (catching) and irradiation should not exceed 5 days. It should be easy to put such a transport plan into effect. It is assumed that the factors that determine and affect the microbial aspect of the shrimp study are identical with those of the lobster tails which have been discussed in Part I (Section 4.4). As in Part I no attempt was made to identify the bacterial species found in the fresh, blanched or irradiated shrimp. The bacteriological tests in this study are used together with other criteria for comparison and assessment of the storage life of shrimp under simulated commercial conditions. In Section 8. 1 and Table XIV it may be seen that the initial number of bacteria varied in the raw material counted after 72 hours incubation at 22°C. The lethal effect of blanching for 2 and 5 min resulted in the surviving bacteria numbering less than 100 bacteria per gram. Both blanching times showed bactericidal efficacy, which was higher in the case of the shrimp than the lobster tails, i. e. reduction of bacterial numbers of about 4.0 log cycles compared with 2.5 log cycles for 2-min blanching of lobster tails. This may be explained in part by the difference in size of the two types of seafoods, and that the shrimp was blanched in shell which includes the head portion and stomach contents (whole, intact animal). This undoubtedly is of significance in evaluating the blanching effect. The bactericidal efficacy of blanching does not appear to be dependent on the initial number of bacteria in shrimp as was suggested for the lobsters.

57 The different action of radiation pasteurization and of blanching have been discussed in Part I (Section 4.4). In essence the same is true here, and will not be dealt with. In Table XV the relative storage life at 0°-l°C is determined. As in Part I the borderline of acceptability for the total bacterial count (TBC) was set at 1 X 106 bacteria per gram. On the whole the storage life of the product is shorter when such a borderline is set at 1 X 106 per gram than that determined by the sensory evaluation score (T-test), the difference being from 1 to 20 days. If the borderline of acceptability judged by total bacterial count (TBC) is raised to 1 X 107 the discrepancy is lessened. In some instances it is equal to the T-test, in other instances it shows great deviation, especially in groups 3 and 4 where combined treatments of blanching and irradiation were used. From a bacteriological viewpoint the treatment of choice for peeled shrimp is to blanch the product for 2 min, and peel and irradiate at a dose level of 1 X 105 rad. The results of storing blanched, unpeeled shrimp for 2 and 5 days and then irradiating at a dose level of IX 105 rad are rather odd. Bacteriologically the stored, unpeeled shrimp showed longer post- irradiation storage life than samples blanched, peeled and irradiated on the same day. Probably this is due to contamination during peeling. As may be seen from Table XIV, bacterial growth had occurred during pre- irradiation storage, but was reduced by irradiation. In connection with the storage of blanched unpeeled shrimp it should be emphasized that the storage temperature should be maintained at 0°-l°C in order to minimize the danger of growth of Cl. botulinum, type E, as this species has been found by Danish workers [22, p. 91] in bottom mud samples in localities where shrimp is caught off Greenland. Generally speaking the sensory evaluation score (T-test) follows the same trends as experienced in the lobster project. However, in this study the shrimp was steamed instead of boiled in a minimum amount of water as with the lobsters. This could be one of the reasons why the panel members rejected the shrimp samples which had received a dose level above 2 X 105 rad as the irradiation off-odour and off-flavour may be more pronounced when samples are steamed. Another reason for the panel members' rejection could be the polyethylene packaging material which imparted a slight off-odour by itself on the unsteamed samples. Quick frozen reference samples containing about 1% sodium chloride were always served along with the test samples. The panel members quite frequently preferred the irradiated shrimp to the reference samples.- Discrepancies in scoring were found on the same day and from day to day as was experienced with the lobster tails. But good agreement existed among the panel members when it came to determining the termination of experiment or the borderline of acceptability. Results in Part I showed that volatile acid number (VAN) determination was of limited value as a quality index for blanched and irradiated lobster tails. In the shrimp study the VAN test was not used but instead the trimethylamine (TMA) test was used for the same purpose. As shown in Table XV and in the figures the TMA determination does not appear to be a reliable index of determining the storage life of blanched and irradiated shrimp.

58 In most instances an increase in the TMA value was observed but poor agreement with the borderline of acceptability as judged by the T-test was noticed. It appears questionable to fix a definite TMA value as a terminal index of storage life for blanched, irradiated shrimp. It was sometimes observed, however, that the TMA did increase substantially after termination of storage life according to the T-test, but since the samples were by that time inedible these TMA values were not included in the figures. In spite of this it is felt that the TMA determination might be used as an approximate indication of the quality of blanched, irradiated shrimp.

10. SUMMARY AND CONCLUSIONS

Reference is made to Table XV for the overall results of the storage life studies on shrimp. The premise and the scope of this study has been explained in Section 9. It should be re-emphasized that conditions in the Icelandic shrimp fishing industry were the governing factors in planning the study. Briefly, the main conclusions reached from this study are as follows:

(i) Blanching of the shrimp for 2 or 5 min is a necessary pre-irradiation treatment in order to delay spoilage before irradiation and furthermore to prevent discolouration of the irradiated product. (ii) The dose level of choice appears to be below 2 X 105 rad, as above that dose undesirable organoleptic changes occur. From the microbial viewpoint a dose level of 2 X 105 rad is, however, desirable as it results in significantly longer storage life, if total bacterial count is used as storage life index. (iii) Blanched, unpeeled shrimp may be kept on ice up to 5 days before peeling and irradiation without affecting post-irradiation storage life significantly. This period should suffice to ship the shrimp from the catching grounds off the northwest coast to the irradiation centre on the southwest coast (see Part I). (iv) Using the optimal combination of treatments, which is considered to be 5-min blanching and irradiation at a dose level of 1 X 105 rad, the storage life of peeled shrimp was found to be about 7 weeks. (v) The criteria used to determine the storage life were total bacterial count (TBC) at 22°C incubation, the sensory evaluation score (T-test) and the trimethylamine (TMA) test. The T-test was used as a basis of comparison for the other two tests. The TBC showed a fairly close agreement with the T-test on untreated or only blanched shrimp but showed significantly a somewhat shorter storage life of the product which had received the combined treatment of blanching and irradiation. The TMA test appears to be unreliable as a terminal index of the storage life of blanched irradiated shrimp, but might possibly be used as quality index as has been established for untreated shrimp.

In this study no attempt has been made to evaluate the economic aspects such as processing costs, costs of distribution and consumer preference. The emphasis was placed on studying the feasible handling of the raw material and storage of the treated product. It is concluded that by blanching

59 the shrimp on board the fishing vessels the shrimp will keep up to 5 days and withstand transport to a proposed irradiation centre. It is also concluded that by using optimal, combined treatment of blanching and irradiation, peeled shrimp has a storage life of almost 6 weeks at 0°-l°C. This should make it possible to ship and sell peeled, irradiated shrimp in foreign markets, for example in Europe.

60 PART III RADURIZATION OF WHOLE COD AND COD FILLETS

11. INTRODUCTION

The third part of the irradiation studies on seafoods deals with the radiation pasteurization of whitefish of which cod, Gadus callarias, is a typical example. Whitefish are a very important part of the Icelandic fishing industry. The total annual whitefish catch is on the average 350-400 thousand metric tons of which 200-250 thousand metric tons are filleted and exported as prime quality quick-frozen fillets and about 20^000 -40 000 metric tons are exported as iced, whole fish. Of the whitefish species the cod is by far the most abundant, and haddock, Melanogrammus aeglifinus, is caught in considerably lesser quantity. As regards price, haddock fetches a higher price than cod and is more sought after on the market. When the experiments on radiation pasteurization of whitefish were started in 1969, they coincided with the cod season which lasts from January to the end of May. The haddock season runs fairly parallel to the cod season, but is more unpredictable. Therefore, cod was chosen for this study. Many reports on radiation pasteurization of whitefish are to be found in the literature [13,16,17,19,25] . The literature reveals that most of the work on low-level irradiation of whitefish has been concerned with fillets. Nevertheless, some authors have worked with whole fish [26,27] . As in the two previous Parts, the major emphasis in this study is on the evaluation of the usefulness of low-level irradiation, in this case of whitefish. An attempt is made to take into consideration special conditions existing in catching methods and fish handling commonly used in the Icelandic fishing industry. Cod is caught in Icelandic waters mainly by three methods: line or inshore fishing by hook and line; by gill netting; and by trawling. Of the catching methods the line fisheries usually produce prime quality fish. The line- caught fish have usually been less than 24 hours out of the water when landed, and are bled while still alive and then iced. Therefore, the fish are most frequently still in rigor when landed. The netted cod is generally of inferior quality to the line fish. The reasons for this are that the nets may stay in the water longer than 24 hours and therefore the fish are often dead or near death when brought on board the fishing vessels, and the bleeding is therefore less efficient. The net fish are of loose texture and are quite often unfit for freezing. The gill netting is pursued to some extent during the main cod season, as it is re- warding in the quantity of fish landed, and enables the fishing vessels to be operated more efficiently. The trawl fisheries are done in deeper waters and the boats stay out longer. The catch is on the average older when landed even though methods of handling on board consist of proper bleeding of the live fish, and thorough icing on board. The quality of the trawl fish are inherently more variable. The most recently caught fish are often comparable with line fish but on the whole most of the catch is much older, even up to 7 or

61 10 days old, but usually not more than 5 days. This is clearly reflected in the quality of the landed fish. The trawl fish are at times exported iced to foreign markets mainly to the United Kingdom and the Federal Republic of Germany. Of the total amount of whitefish landed in Iceland about half is quick-frozen as fillets, the remainder being salted or dried (stockfish). Upon landing in the evening or during the night, the fish are washed and iced. The following day the processing of the fish begins by washing, machine filleting, machine skinning and manual trimming and packaging. The packaged fish are then quick- frozen in contact plate-freezers. It is clear that the method of catching determines the quality of the fish landed and the quality of the product. The aim of this study was to attempt to elucidate the importance of the quality of the raw cod as it prevails in Iceland for the post-irradiation storage life of cod fillets and whole, eviscerated cod. In processing cod fillets before irradiation it is assumed that the same methods of handling and preparation would be adhered to as is customary in processing quick-frozen fillets. This approach was used in that part of this study which deals with radiation pasteurization of cod fillets. Most of the raw material was of commercial origin, obtained from freezing plants after trimming and ready to be packed and frozen. In two experiments cod was filleted in the laboratory under controlled conditions to see the effect of sanitation in handling and preparation. The radiation pasteurization of whole cod caught by net and line was undertaken because a considerable amount of iced, whole fish is exported and sold as such, in addition to the trawl fish referred to above. Further extension of the storage life of this fish is of great importance to this branch of the Icelandic fish trade. In some of the countries in which the fish is marketed the customers prefer iced, whole fish to quick-frozen fish.

12. MATERIALS AND METHODS

As regards dosimetry, packaging, storage and analytical methods for assessment of quality and determining the storage life reference is made to previous reports with the exception of the sensory evaluation which will be described in the appropriate section [28] .

12.1. Raw material, origin and preparation

The raw material for the study of radiation pasteurization of cod fillets was in most instances obtained from the same commercial freezing plant. The cod fillets were of three different types or origin as determined by methods of catching: by line fishing, gill net fishing and trawl fishing. The fillets were obtained from the freezing plants after skinning and trimming and were packaged before irradiation in the laboratory. The fillets were of fish landed the night before and had received conventional, commercial handling before being brought to the laboratory. In the freezing plants and in the laboratory the three types of fillets (line, net and trawl caught) received the same handling but were in different state of freshness and quality resulting from the different methods of catching.

62 The whole cod was obtained directly from fishing vessels at the time of landing and was brought to the laboratory where necessary preparations for irradiation were made. Owing to the limited size of the irradiation containers, only fish of medium size or smaller could be selected. The fish were beheaded (gutting is done on board the fishing vessels), the tail fin cut off, washed and packaged. The whole cod was of two types, i.e. line-caught and net-caught fish. In the laboratory two experiments were made on fillets which were hand-cut from unirradiated line cod. In one of the experiments particular attention was given to sanitation when the fillets were hand-cut, skinned and packaged, whereas in the other experiment no special effort was made in this respect and this could be considered to simulate the normal hand- cut filleting operation in the freezing plants. Fillets of stored, irradiated whole cod were hand-cut by the normal procedure, without any special sanitary precautions, before re-irradiation of the fillets. For information on aseptic sampling for bacteriological tests see Section 12.7.

12.2. Packaging

The packaging material used in this study was polyethylene bags of 0.07-mm thickness. About 500-700 g of fillets consisting of two or three pieces were packaged individually; the bags were then heat-sealed with the minimum possible amount of air (not vacuum packed). Whole cod was packed individually in two polyethylene bags, one over the other, the bags were not heat-sealed but closed by tying knots at each opening. One preliminary experiment on the effect of packaging material on the organoleptic scoring of irradiated cod was performed (see Section 13. 1).

12.3. Stora ge

Fillet samples were stored at either 0°-l°C or 5°-6°C. All whole cod samples were stored at 0°-l°C.

12.4. Irradiation dose

Dose levels of irradiation commonly used for whitefish (cod) fillets range from 1. 0 X 105 to 2. 5 X 105 rad [25, 29] . For radiation pasteurization of cod fillets the dose levels tested were 0. 5 X 105 , 1. 0 X 10 5, 2. 0 X 105 and 3. 0 X 10 5 rad. As may be seen from Table XVI, fillets irradiated at a dose level of 3.0 X 105 rad showed distinctly lower organoleptic scores than those irradiated at the lower doses. The dose level chosen in this study for radiation pasteurization of cod fillets was 2.0 X 105 rad. The dose level chosen for irradiation of whole cod was 0.5X 105 rad. In the literature, dose levels of 0. 5 X 105 rad and 1. 0 X 105 rad [10,26] have been used for whole whitefish (cod, haddock and English sole). A dose level of 0.5 X 10® rad was used to irradiate whole cod. Re-irradiation of fillets from pre-irradiated whole cod was done at a dose level of 1.5 X 105 rad.

63 TABLE XVI. PRELIMINARY TESTS ON THE INFLUENCE OF TWO TYPES OF PACKAGING MATERIAL AND DIFFERENT DOSE LEVELS OF IRRADIATION ON SENSORY EVALUATION SCORES OF RAW COD FILLETS, JUDGED BY 8 PANEL MEMBERS ON A 5-POINT PREFERENCE SCALE

Packaging material Dose levels of Mean score for irradiation Plastic bags Cans, 1 lb each dose level (rad) average score average score

Control 0 4.05 4.00 4.03

0.5 x 105 3.95 3.80 3.88 1.0 x 10s 3.60 3.50 3.55 s 2.0 x 10 3.40 3.55 3.48 3.0 x 10s 2.60 2.80 2.70

Sum average 3.52 3.53

Dosimetric determinations were performed as described in Part II, Section 7.1.5, with cod fillets as the medium. The max./min. ratio was found to be 1.40. The average dose rate was about 420 rad/sec.

12.5. Sensory evaluation (O-test)

The sensory evaluation used in this study is based on the technique used at the Technological Laboratory, Bureau of Commercial Fisheries, Gloucester [28] . Details of the procedure were obtained during a visit to the Gloucester Laboratory. The technique is handy as little preparation is needed and appears to give reliable results on the limit of acceptability. This sensory evaluation is based on the odour of the raw (uncooked) product, the O-test. The actual scoring involved estimation by the panel members of the age (in days) of the product compared with reference samples of known age. The odour test was performed as follows: the samples, never more than four at a time, were removed along with the reference samples from cold storage. A large, representative piece of fillet (300-400 g) was removed from the bag and put into a 1-litre glass container fitted with a lid and left to stand at room temperature for li to 2 h before sensory evaluation was undertaken by the panel members. The temperature of the samples was then in most cases about or above 10°C. The panel members were then requested to record their estimation of age (in days) of the test samples, the emphasis being placed on their decision as to whether the samples were edible or not. The date of rejection of each sample was defined as that date on which the panelists unanimously, or with not more than one exception, agreed that it was inedible. The physical appearance of the samples was not included in the sensory scoring but served only as a guideline. The main emphasis was on determining the day when the odour indicated that the fish was inedible.

64 The sensory evaluation is referred to in the text, tables and figures as the O-test.

12.6. Chemical tests

The chemical tests used for assessment of quality and determination of storage life were the volatile acid number determination (VAN) and the trimethylamine determination (TMA). The description of the procedure for determining VAN is given in Part I, Section 2.2.2. The description of the procedure for determining TMA is given in Part II, Section 7.2.3.

12.7.. Bacteriological determination (TBC)

The methods of sampling used for bacteriological testing were as follows: Commercial cod fillets received at the laboratory were sampled by cutting two small pieces of each fillet by sterile knives and forceps. The pieces of fillets were then pooled, mixed and ground in a sterile meat grinder, and 44 g aliquots were weighed out into a sterile Waring blender jar. Samples of stored cod fillets packed in polyethylene bags were sampled aseptically by removing about half of each bag1 s contents along with the "drip" from the fillets and were then mixed in a sterile meat grinder, and 44 g aliquots weighed into a sterile Waring blender jar. Whole cod was sampled in two ways, firstly, as "under the skin" samples and, secondly, as fillets after laboratory filleting. The samples from the whole cod from underneath the skin were taken in the following manner: the cod was placed on a clean cutting table on a . piece of paper, an incision was then made in the skin with a sterile filleting knife along the back, across the tail fin section, along the ventral side around thè belly flaps to the neck region where a cross incision was made. During this operation care was taken to allow the knife just to pierce the skin so as to keep contamination of the underlying tissue to a minimum. With a sterile páir of pliers in one hand and holding the tail end firmly, the skin was ripped away from the flesh in one motion. The exposed flesh was then excised by cutting lengthwise about 2 cm from the periphery all the way to the backbone, the excision being done with sterile knives and forceps. The flesh thus collected was then ground in a sterile meat grinder and an aliquot sample of 44 g weighed into a sterile Waring blender jar. When cod fillets were prepared in the laboratory, the bacteriological- sampling procedure was the same as described above for sampling commercial cod fillets. The 44 g aliquots of cod in the Waring blender jar were then mixed by adding 400 ml of sterile saline dilution solution and mixing (spinning) carried out for 2 min at medium speed. Appropriate serial dilutions into saline solution were then done and plated on Petri plates. The culture medium used was that of Liston and Matches [10] with the modification that 0.25% dextrose was added to the medium. All plates were incubated at 22°C for 72 h at which time they were counted with the aid of a Quebec colony counter .

65 13. RESULTS

13.1. Results of O-test

A description of the sensory evaluation (O-test) used in this study is given in Section 12.5. It was found necessary, however, to train the panel in evaluating raw, unirradiated and irradiated cod by this technique. For this purpose a group of panel members was first trained in estimating the age of unirradiated cod samples by the odour of the raw product! This training period lasted for about two months. The panel members were then trained to become accustomed to the irradiation odour, and those who showed the best ability to characterize the degree of spoilage in spite of the irradiation odour were placed on the panel. At the initial stages of the experiments of cod irradiation, the choice of packaging material was in doubt. It was therefore decided to use two readily available packaging materials to test the effect of the packaging material, as such, on the odour-evaluation score of cod irradiated at different dose levels. The materials in question were polyethylene bags of 0.07-mm thickness and 1-lb enamelled cans (see Part I, Section 2.1.2). Polyethylene has already been approved by the US Food and Drug Administra- tion as a packaging material for radiopasteurized food [30] , but it is considered to impart some off-odours to the food, whereas enamelled cans do not. The results of this test are presented in Table XVI. Eight panel members made their judgements on a 5-point scale. As may be seen from Table XVI, no significant difference was observed between the two types of packaging materials. It was therefore decided to use the plastic bags which were much more convenient and less expensive. It may also be seen that the dose level of 3 X 105 rad resulted in considerably lower scores, whereas small difference was detected between 1 X 105 and 2X10 rad and 0 and 0. 5 X 105 rad respectively. For this reason, the dose level of 3 X 105 rad was abandoned, even though, as may be seen from Table XVII, this dose showed the best bactericidal efficacy. Tables XXI- XXVIII show the average age determination of the samples, in days, estimated by the panel members, using the O-test technique. The O-test values signifying inedibility or date of rejection are identified in the tables by suffixing the letter "r" to the panel members' score values on that day. Results of the O-test during storage of cod fillets are to be found in Tables XVIII, XXIII-XXVI.- As indicated in Section 12. 1, the raw material were fillets of line, net and trawl-caught cod. Results show that for unirradiated line cod fillets kept at 0°-l°C the date of rejection was 7 days, whereas net cod and trawl cod kept for 5 days. When the same types of cod fillets were stored at 5°-6°C, the date of rejection was in all instances 3-4 days. For the cod fillets irradiated at a dose level of 2 X 105 rad and kept at 0°-l°C the date of rejection for line cod was 24 days, 20 days for net cod and 19 days for trawl cod. When kept at 5°-6°C the line cod was rejected after 13 days and net and trawl cod after 11 days. In Table XVIII comparison is made between 1 X 105 and 2 X 10s rad dose levels at two storage temperatures. Results are self explanatory. A summary table. Table XIX, compares the end of storage life of three different types of fillets, under different storage conditions unirradiated and irradiated as determined by the three test criteria.

66 TABLE XVII. COMPARISON OF INITIAL AND SURVIVING LOG NUMBER OF BACTERIA IN FILLETS OF LINE, NET AND TRAWL COD AFTER IRRADIATION AT 1.0 X 105 AND 2.0 X 105 RAD; INCUBATION TEMPERATURE 22°С

1.0 x 10s rad 2.0 x 105 rad Initial Raw material log no. Log no. of Log cycle Log no. of Log cycle fillets 0 rad survivors reduction survivors reduction

A. Line cod

Trial 1 5.4 3.4 2.0

" 2 6.0 4.5 1.5 3.4 2.6

" 3 5.7 3.4 2.3

B. Net cod

5.8 2.9 2.9

C. Trawl coda

Trial 1 6.0 3.6 2.4

" 2 6.0 3.5 2.5 2.7 3.3

" 3 6.1 3.9 2.2

a One trial using 3.0 x 105 rad showed 4.3 log cycle reduction.

The O-test on the whole cod was done in the same manner as on the cod fillets. In Table XX are shown the comparative results of O-tests of fillets of whole cod caught by line and net. For the line cod irradiated whole at 0.5 X 105 rad, the date of rejection was 21 days compared with 12 days for the unirradiated control. For the net cod the rejection times were 13 days for samples irradiated with 0.5 X 105 rad and 6 days for the unirradiated samples. It should be pointed out that the net cod was of far more inferior material quality than the line cod as the former showed an O-test score of 5 at 0 days compared with 1.5 for line cod. Furthermore, the net cod showed distinct discolouration of the flesh, along the backbone and in the belly flaps due to improper bleeding. The fillets from the whole net cod which had been stored for 6 days were upon re-irradiation with 1.5 X 105 rad judged inedible by the majority of the panel because of extreme off-odours. Fillets from whole line cod irradiated with 0. 5 X 10s rad and stored for 3, 7 and 12 days, and then cut and re-irradiated with 1. 5 X 105 rad, were judged inedible after 18-20 days of fillet storage. Total storage life was 23, 27 and 30 days respectively (see Table XXI and Fig. 20). Table XXII shows the comparative results of whole irradiated (0.5X 105rad) line cod versus commercial- and laboratory-prepared line cod fillets irradiated at a dose level of 2 X 105 rad. By using extra care (sanitation) in filleting the rejection time was 33 days compared with 24 days for commercial line cod fillets. Filleting by hand in which no precautions were taken whatsoever showed drastically shorter storage life, only 19 days.

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68 TABLE XIX. SUMMARY OF STORAGE LIFE IN DAYS OF COD FILLETS UNIRRADIATED AND IRRADIATED AT A DOSE LEVEL OF 2 X 105 RAD; STORAGE TEMPERATURES 0° - Io С AND 5° - 6° C; ASSESSMENT TESTS BY ODOUR EVALUATION (O-TEST), TRIMETHYLAMINE (TMA) AND TOTAL BACTERIAL COUNT AT 22° С (TBC)

0-test Dose level Storage Origin of time of TMA TBC of temp. cod fillets rejection a5.mg N/100 g alo7 irradiation (days) (days) (days)

5° - 6°C 0 rad Line cod 3 2 2

Net cod 4 2 2

Trawl cod 4 2 2

0°-l°C Line cod 7 6 6

Net cod 5 4 5

Trawl cod 5 4 5

5° - 6°C 2 x 105 rad Line cod 13 11 8

Net cod 11 9 7

Trawl cod 11 8 6

0° - rc 2 x 105 rad Line cod 24 22 14

Net cod 20 16 12

Trawl cod 19 17 11

0°-l°C 2 x 10s rad Line cod special lab. filleting 33 31 26

13.2. Results of chemical tests (VAN and TMA)

Results of the volatile acid number determinations, VAN, showed trends similar to those found in the storage studies on lobster tails (see Part I). Thus the unirradiated control samples showed a distinct increase in volatile acids during storage which appeared to correlate with the other storage test criteria. The same appeared to be valid for whole cod irradiated with 0.5 X 105 rad (see Table XX). For the cod fillets irradiated at higher dose levels (IX 105, 1.5 X 10s or 2 X 105 rad) only two or three individual samples showed some increase in volatile acid number. The trimethylamine values for stored cod fillets of the three different types,line-caught, net-caught and trawl-caught cod, are to be found in Tables XVIII, XXIII-XXVI and Figs 21 and 22. As may be seen, the TMA increased substantially in all instances but a definite delayed rise in TMA

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71 FIG.20. Comparison of storage life of whole line cod, pre-irradiated at a dose level of 0.5 X 10srad and fillets from the whole cod re-irradiated at a dose level of 1. 5 X 105 rad. The whole cod was stored 3, 7 and 12 days before filleting. Storage life determined by trimethylamine determination (TMA). Storage temperature 0°-l°C.

was observed for the irradiated cod fillets. The rise in TMA occurred in most instances shortly before the date of rejection. In this study the TMA value of 5 mg N TMA./l00gfish flesh, a figure which has been suggested as a threshold value for fish fit for marketing [31] , was used as the borderline value of acceptability. Table XIX shows a close correlation between the O-test time of rejection and the TMA value of 5 mg N TMA/100 g, but this value was reached 1 to 4 days earlier than the rejection date. Of the three types of raw material, the fillets of the trawl cod showed TMA values above 1 mg N ТМА/lOOg whereas the line and the net cod fillets had an initial value of about 0. 5 mg N TMA/100 g. As regards the whole cod, both the unirradiated control samples and the samples irradiated with 0. 5 X 105 rad showed a rather sharp rise in the TMA curve (see Fig. 23) but the irradiated samples showed delayed response. The cod fillets cut from whole, irradiated cod and then re-irradiated showed an expected increase in TMA upon storage. The borderline of 5 mg N TMA./I00g, however, seemed to be reached here on or even just after the date of rejection. The laboratory-prepared cod fillets with and without special care in handling (sanitation) showed similar trends in TMA production as the commercially prepared fillets (see Fig. 24).

Text continued on page 82

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25 26 •AYS OF STORAGE FIG. 21. Comparison of storage life of unirradiated (controls) and irradiated (dose level 2 X 10s rad) cod fillets caught by line, net and trawl as determined by trimethylamine determination (TMA). Storage temperature 0°-l°C.

FIG. 22. Comparison of storage life of unirradiated (controls) and irradiated (dose level 2 X 105 rad) cod fillets caught by line, net and trawl as determined by trimethylamine determination (TMA). Storage temperature 5°-6°C.

78 FIG. 23. Comparison of storage life of whole line and net cod unirradiated (controls) and irradiated at a dose level of 0. 5 X 10s rad as determined by trimethylamine determination (TMA). Storage temperature 0°-l°C.

FIG. 24. Comparison of storage life of whole line cod irradiated at a dose level of 0. 5 X 10s rad with commercial and laboratory-prepared line cod fillets irradiated at a dose level of 2 X 105 rad as determined by trimethylamine determination (TMA). Storage temperature 0°-l°C.

79 FIG. 25. Comparison of storage life of unirradiated (controls) and irradiated (dose level 2 X 105 rad) cod fillets caught by line, net and trawl as determined by total bacterial count (TBC) at 22°C. Storage temperature 0°-l°C.

FIG. 26. Comparison of storage life of unirradiated (controls) and irradiated (dose level 2 X 105 rad) cod fillets caught by line, net and trawl as determined by total bacterial count (TBC) at 22°c. Storage temperature 5°-6°C.

80 TABLE XXVII. LINE COD: THE EFFECT OF HANDLING AND STORAGE VARIABLES ON THE TOTAL BACTERIAL COUNT AT 22°С OF WHOLE COD AND FILLETS, USING LOW-LEVEL IRRADIATION; STORAGE TEMPERATURE 0° - Io С

Log no. of Log cycle Handling, storage and irradiation variables survivors reduction

A. START OF EXPERIMENT 1. Whole cod, aseptic sampling 0 rad 1.0 2. Whole cod, aseptic sampling 0.5 x 105 rad 0 1.0 3. Fillets from whole cod, lab. filleting 0 rad 5.0 4. Fillets from whole cod, lab. filleting 2.0 x 10s rad 3.1 1.9 5. Fillets from whole cod, special lab. filleting 0 rad 3.2 6. Fillets from whole cod, special lab. filleting 2.0 x 10s rad 1.0 2.2

B. AFTER STORAGE FOR 3 DAYS 7. Whole cod (see A.l), aseptic sampling 0 rad 2.1

8. Whole cod (see A. 2), aseptic sampling 0.5 x 10s rad 0 9. Fillets from whole cod (see A. 2), lab. filleting 0.5 x 10s rad 3.0 10. Fillets from whole cod (see A.2), 0.5 x 10s rad lab. filleting and re-irradiation 1.5 x 10s rad 1.0 2.0

C. AFTER STORAGE FOR 7 DAYS 11. Fillets from whole cod (see. A.2), lab. filleting 0.5 x 10s rad 4.9 12. Fillets from whole cod (see A.2), 0.5 x 105 rad lab. filleting and re-irradiation 1.5 x 105 rad 4.4 0.5

D. AFTER STORAGE FOR 12 DAYS 13. Whole cod (see A. 1), aseptic sampling 0 rad 5.2 14. Whole cod (see A.2), aseptic sampling 0.5 x 10s rad 3.5 15. Fillets from whole cod (see A.2), lab. filleting 0.5 x 10s rad 5.2 16. Fillets from whole cod (see A.2), 0.5 x 105 rad lab. filleting and re-irradiation 1.5 x 105 rad 3.8 1.4 17. Fillets from whole cod (see A.4), lab. filleting 2.0 x 105 rad 6.2 18. Fillets from whole cod (see A.6), special lab, filleting 2.0 x 10s rad 3.6

81 13.3. Bacteriological determination. (TBC)

In this study the number of initial and surviving bacteria after irradiation at different dose levels of irradiation before final storage was determined in a similar manner to that indicated in Part I (Section 3. 2) and Part II (Section 8.1). The bactericidal efficacy of pasteurization doses of irradiation of cod fillets of line, net and trawl cod is shown in Table XVII. The log number of initial bacteria present in all types of cod fillets per gram ranged from 5.4 to 6.1, averaging 5.9. Fillets of line cod showed on an average a 1. 75 log cycle reduction in bacterial numbers when irradiated at a dose level of 1.0X 105 rad but 2.45 log cycles when irradiated at 2.0 X 105 rad. Fillets of net cod showed a 2. 9 log cycle reduction in bacterial numbers when irradiated at a dose level of 2. 0 X 10s rad. Fillets of trawl cod showed an average 2.4 log cycle reduction at l.'OX 10s rad and 3.3 log cycle reduction in bacterial numbers when irradiated at a dose level of 2.0 X 105 rad. The results of the total bacterial counts of fillets of line cod, net cod and trawl cod stored at 0°-l°C and 5°-6°C are shown in Tables XVIII, XXIII-XXVI. In Table XIX is shown the comparative storage life in days. Figures 25 and 26 present graphically the increase in bacterial counts during storage. The borderline of total bacterial count acceptability is set in this study at 1.0 X 107 bacteria per gram after incubation at 22°C. From the tables and the figures it-may be seen that irradiation at a dose level of 2.0 X 105 rad prolongs the storage life at 0°-l°C by a factor of approximately two compared with unirradiated samples. The irradiated fillets of line cod showed the longest storage life. Net cod fillets and trawl cod fillets showed 2 days and 3 days shorter life, respectively, than the line cod fillets. Fillets of line cod prepared in the laboratory by special care in sanitation and then irradiated with 2. 0 X 105 rad dose showed about twice as long a storage life as the commercial fillets of line cod, irradiated with the same dose. Storage life of unirradiated cod fillets stored at 5°-6°C is about one third to one half of that of the fillets stored at 0°-l°C. Storage life at 5°-6°C of cod fillets irradiated at 2.0 X 105 rad is about one half of that of the fillets stored at 0°-l°C. As may be seen from Table XIX the storage life in days of the unirradiated cod fillets using 1 X 101 bacteria per gram as the limit is in good agreement with other assessment tests, but shows a far shorter storage life than the other assessment tests for the irradiated cod fillet samples. Similar results were obtained in earlier studies (see Parts I and II). The data of the bacteriological tests on whole cod and fillets prepared in the laboratory are presented in Tables XXVII and XXVIII and Figs 27-29. The type of raw material used in these experiments was line cod and net cod. The data collected deal not only with the bactericidal efficacy of low- level irradiation on whole cod and storage life determination of whole cod and fillets made intermittently from stored whole cod, but takes also into consideration some of the handling variables during filleting which may affect storage life.

82 TABLE XXVIII. NET COD: THE EFFECT OF HANDLING AND STORAGE VARIABLES ON THE TOTAL BACTERIAL COUNT AT 22° С OF WHOLE COD AND FILLETS, USING LOW-LEVEL IRRADIATION; STORAGE TEMPERATURE 0° - Io С

Log no. of Log cycle Handling, storage and irradiation variables survivors reduction

A. START OF EXPERIMENT

1. Whole cod, aseptic sampling 0 rad 1.6

2. Whole cod, aseptic sampling 0.5 x 10s rad 1.0 0.6

3. Fillets from whole cod, lab. filleting 0 rad 3.7

4. Fillets from whole cod, lab. filleting 2.0 x 10s rad 2.6 1.1

B. AFTER STORAGE FOR 6 DAYS

5. Whole cod (see A. 1), aseptic sampling 0. rad 4.5

6. Whole cod (see A.2), aseptic sampling 0.5 x 105 rad 4.7

7. Fillets from whole cod (see A.2), 0.5 x 10s rad special lab. filleting 4.6

8. Fillets from whole cod (see A.2), 0.5 x 105 rad special lab. filleting and re-irradiation 1.5 x 105 rad 2.3 2.3

9. Fillets from whole cod (see A.4), lab. filleting 2.0 x 105 rad 2.9

The bactericidal efficacy of 0.5 X 10s rad irradiation showed a 0.6 and 1.0 log cycle reduction of bacterial numbers for net cod and line cod respectively. In Table XXVII the results of the bacterial counts during the experiment on whole line cod are given. In Section A of Table XXVII are results of bacterial counts taken on the day the experiment was begun. Results show that the cod tissue contains only 10 bacteria per gram which is reduced to 0 after 0.5 X 105 rad irradiation. These bacterial counts were obtained from samples taken aseptically. Fillets made from the same cod, on the same day, using no special care (sanitation) during the filleting operation showed that the contamination was up to about 1 X 105 bacteria per gram. After irradiating these fillets at a dose level of 2. 0 X Í05 rad, a 1. 9 log cycle reduction in the number of bacteria was found and the number of survivors was 1.3 X 103 per gram. By comparison filleting with special care in sanitation showed a count of 1.4 X 103 and 1.0 X 101 bacteria per gram after irradiation by 2. 0 X 10s rad.

83 FIG. 27. Comparison of storage life of whole line and net cod unirradiated (controls) and irradiated at a dose level of 0. 5 X 105 rad as determined by total bacterial count (TBC) at 22°C. Storage temperature O'-l'C.

FIG. 28. Comparison of storage life of whole line cod, pre-irradiated at a dose level of 0. 5 x 105 rad and fillets from the whole cod re-irradiated at a dose level of 1. 5 X 10srad. The whole cod was stored 3,7 and 12 days before filleting. Storage life determined by total bacterial count (TBC) at 22°C. Storage temperature 0°-l°C.

84 FIG. 29. Comparison of storage life of whole line cod irradiated at a dose level of 0. 5 X 105 rad with commercial and laboratory-prepared line cod fillets irradiated at a dose level of 2 X 105 rad as determined by total bacterial count (TBC) at 22°C. Storage temperature 0°-l°C.

In Table XXVII the events are given in chronological order for up to 12 days storage of whole line cod and the effect is shown of storage and variation in handling. The effect of care in handling is clearly borne out by consulting Section D in Table XXVII and comparing items 13-18. Thus if items 17 and 18 are compared, i.e. the bacterial counts of the fillets irradiated at the start of the experiment with the same dose levels of 2.0 X 105 rad, the one lot where special sanitary care was taken during filleting has 4.0 X 103 versus 1.4 X 106 bacteria per gram when no special sanitary care was used in filleting after 12 days storage at 0°-l°C. Fillets made from pre-irradiated cod (0.5 X 105 rad) at the start of the experiment contained after 12 days storage 1.4 X 105 bacteria per gram and those which were at the 12th day re-irradiated with 1. 5 X 105 rad showed 6.OX 103 bacteria per gram after re-irradiation, a result similar to that of fillets irradiated at 0 days with 2. OX 105 rad. In Table XXVIII are the results of the effect of handling during storage on the total bacterial counts of whole net cod and net cod fillets. The effect of sanitation in the filleting operation is again evident. Table XX and Fig. 27 show the total bacterial counts of whole line and net cod. It may be seen that the line cod, both unirradiated and irradiated at a dose level of 0.5 X 105 rad, showed significantly longer storage life than net cod. Irradiated net cod showed a similar storage life as unirradiated line cod. In this experiment the total bacterial count results (aseptically sampled) indicated longer storage life than the other assessment tests (Otest and TMA); this is contrary to what was experienced with the cod fillets.

85 In Table XXI and Fig. 28 it may be seen that the fillets from pre- irradiated whole line cod follow the same pattern as found with the commercial fillets (see above). Re-irradiation appears therefore to extend the total storage life. In Table XXII and Fig. 29, where comparison is made between commercial and laboratory-prepared fillets and whole, irradiated line cod (aseptic sampling) and specially prepared laboratory fillets, it may be seen that the former behaved bacteriologically similarly, whereas the latter showed significantly longer storage life. Here again the effect of care in handling (sanitation) is evident.

14. DISCUSSION

In the introduction (Section 11), it was stated that whitefish — cod and haddock — are caught mainly by three methods, by line, by gill-netting and by trawling. Of the three methods of catching that by line gives on the whole the best quality fish, the trawl fisheries give fish of more variable quality whereas the net fisheries yield fish of the least good quality of the three methods. The aim of this study was to investigate the effect of the quality of the cod raw material on the quality of the irradiated product and its effect on the extension of storage life. This is known to affect the quality of iced and frozen fish. The difference in quality between line-caught fish and net- caught fish lies in the catching method rather than in the time elapsed between capture and the beginning of processing. For the trawl fish, however, the time of storage on ice is the limiting factor for quality. The commercial fillets used in this study were of known past history when received at the laboratory from the freezing plant. This includes information on the method and time of catching and the period of storage before and after landing. To evaluate the initial quality and the quality during storage, four test criteria were used as outlined in Sections 12.5, 12.6, and 12.7. For sensory evaluation the O-test method developed by Learson and Ronsivalli [2 8] was used in this study, but modified so that emphasis was placed on determining the time of rejection (see Section 13. 1). The results in this study are similar to those obtained by Learson and Ronsivalli except that these workers found that the time of rejection for fresh fish was 10-12 days whereas in this study the same time was found to be 9-10 days. This may in part be attributed to the difference in preference by the two respective panels. The O-test is a convenient and reliable method of evaluating the quality of fresh or iced whitefish which is of great value to a fish exporting country. Even though the panel members were trained to evaluate irradiated fish, fluctuations and discrepancies in age estimation were found. Lack of long experience in evaluating irradiated foods may account for this. For this reason no figures were prepared showing the O-test develop- ment during storage but the end of storage life is expressed as the time of rejection when the panel members unanimously, or with not more than one exception, agreed that the samples were inedible. Of the chemical tests, the VAN proved to be of limited value for irradiated cod fillets. This is contrary to Spinelli1 s findings [8] which

86 showed that the VAN correlated fairly well with sensory evaluation tests. As regards the TMA determination, Spinelli states "that the utility of TMA as a quality index is applicable only to fish irradiated below 0.2 Mrad" . Spinelli states further: "TMA depending on dose can also be related to sensory changes". Nevertheless Spinelli attaches less significance to TMA than to VAN, which is contrary to what was found in this study (see also Parts I and II), as in no instance was a correlation found between the VAN and the other three test criteria for samples irradiated with 2X 105 rad. On the other hand,. TMA values showed fairly good agreement (see tables and figures) with the O-test and the TBC, lying in between the two tests but closer to the O-test. A borderline TMA value of 5 mgN TMA./100 g of fish has been suggested by Castell et al. [31] . This TMA value was usually reached one or two days before the date of rejection determined by the O-test, for both irradiated and non-irradiated samples (see Table XIX). Some discrepancy and inconsistency were found in the TMA values and this made the drawing of the TMA figures more difficult. It is, however, felt that the TMA value is a relatively reliable test criteria for storage life determination of irradiated cod. More intensive research work will be needed to determine if the borderline value of 5 mgN TMA/100 g fish is valid for irradiated cod. In the bacterial testing of the cod fillets it was found that the bactericidal efficacy of 1. 0 X 105 rad dose level was somewhat lower than expected and the same holds true for 2. OX 105 rad, whereas one trial with 3.0 X 105 rad showed good reduction in bacterial numbers (see Table XVII). This is not readily explainable but might suggest more resistant bacterial flora originating from the environment in the freezing plants and from that of the laboratory after filleting of the fish. This low bactericidal efficacy undoubtedly affects the total storage life of the fillets as the bacterial borderline of acceptability would be reached earlier. It has been suggested in the literature [8, 10,32] that the borderline of acceptability for bacterial counts of irradiated fish should be set at a higher scale than 1 X 106 bacteria per gram which is the limit set for unirradiated fish. Bacterial counts of 1 X 107 bacteria per gram or one or two log cycles higher have been proposed. In the previous studies 1 X 10® and/or 1 X 107 bacteria per gram were used, but in this report 1 X 107 bacteria per gram are used exclusively. Nevertheless, the irradiated (2. OX 105 rad) cod fillets reached a count of 1 X 107 per gram at an earlier time than the O-test time of rejection indicated (see Table XIX). The unirradiated fillets kept at 0°-l°C showed a close agreement between the three test criteria. With the irradiated fillets the relationship between the TBC, TMA and O-test was such that the borderline of acceptability was reached earliest by TBC, then by TMA and finally by the O-test. For the irradiated fillets it was shown that storage life determined by the O-test, TMA and TBC was dose-dependent. The dose level of 3.0 X 105 rad proved to be unacceptable but only slight preference was registered by the panel members between 1.0 X 105 and 2.0 X 105 rad. Contrary to proposals made by US workers and reported by Holston [32] , that 1.0 X 105 rad radiation could be used for a minimum justifiable storage life extension, it is felt that for Icelandic conditions a longer storage life extension would be necessary in order to allow irradiated fish products to be shipped to foreign markets.

87 By using 2.0 X 105 rad a few days longer storage life was obtained than by 1. 0 X 105 rad. This was the chief reason for using 2. 0 X 105 rad dose level for irradiation of cod fillets in this study. A storage temperature of 5°-6°C (refrigeration) gave about one half of the storage life at 0°-l°C. By irradiating the three types of cod fillets by 2.0 X 105 rad the storage life at 0°-l°C was extended 3-4 times beyond that of the control. The storage life of the line cod fillets lasted up to 24 days whereas, that of the net and the trawl cod fillets was 19 and 20 days respectively. The storage life of the best fillets (line cod fillets) is shorter than what has been reported by Ampola et al. [25] for cod fillets irradiated at 1.5 X 105 rad. By using special care in sanitation and handling of line cod during filleting (see Section 12. 1 and Table XIX), a storage life of 33 days was obtained, which correlates with the results of Ampola et al. This suggests the importance of good housekeeping and sanitation during processing of fish to be pasteurized by low-level radiation. This, of course, affected the control samples also by giving them a storage life of 5-7 days compared with 9-10 days which was found during the training period of the O-test panel, when line cod was used. The importance of sanitation will be discussed below. The fillets of line cod showed significantly longer storage life than those of net and trawl cod. As has been indicated in the introduction, the trawl boats may stay out up to 10 days but certain trawl boats stay out only five days, bringing in fish from four to less than one-day-old. The trawl cod used in this study was from a trawl boat which had been fishing for four days and the samples of cod used for filleting were taken at random. The variation in age of the trawl cod catch may explain the shorter storage life before and after irradiation. Trawl fish (cod) should, therefore be landed daily for these purposes. The net cod is usually landed within 24 hours after capture. In many instances the cod is dead or near death at the time of capture and bleeding is less effective. In this study the net cod was dead at the time of capture ("dead bled") and used as such on purpose. Net cod, dead at time of capture, has an unappealing appearance, the flesh and belly flaps being of reddish colour because of poor bleeding, and the flesh is of loose texture. Fillets cut from this type of cod would for this reason alone be considered undesirable for radiation preservation. The shorter storage life of net cod compared with line cod might be due to the ineffective bleeding of the net-caught cod which results in easier bacterial invasion and more enzymatic activity in the flesh. The low-level irradiation of whole, eviscerated cod was tested because a considerable quantity of iced, whole cod is exported to European countries. In the two major importing countries, the United Kingdom and the Federal Republic of Germany, many customers prefer to buy fresh whole fish to fillets and either to frozen. The dose level of 0. 5 X 105 rad chosen in this study is quite likely the lowest effective dose level for significant storage life extension of whole fish. This dose level has been suggested and used by US workers [10 and 26] but is intended for pre-irradiation of whole fish followed by re-irradiation of the fillets at higher dose levels (1.0X 105 to 2. OX 105 rad). Even though it would have been desirable to test higher dose levels for the irradiation of whole cod, the intention was also to test the effect of re-

88 irradiating the fillets cut from the pre-irradiated whole cod. It was desired that the total dose should not exceed 2.0 X 105 rad. It was also concluded from the O-tests, Table XVI, that the dose level of 0. 5 X 105 rad would not cause radiation off-odours to even the most fastidious fish eaters. Testing of more, or higher, dose levels to pinpoint the optimum dose level(s) for maximum storage life extension without affecting the quality, odour and flavour, would have been too time-consuming and complex within the frame and scope of this study. In order to emphasize the importance of the raw material the quality of whole line and net cod was compared. As pointed out earlier in this report, the method of catching rather than the age after time of capture determines the quality of the fish landed. Table XX shows that the whole net cod, both unirradiated and irradiated, had a storage life which was only about a half of that of the line cod. Further- more, the net cod gave from the beginning a much worse O-test rating and the appearance (not included in the O-test) was far inferior to that of the line cod. Net cod fillets, re-irradiated with 1.5 X 105 rad after six days of storage as whole, pre-irradiated cod, were rejected from the beginning by the panel. From this it was concluded that net-caught whole cod is unsuitable for radiation preservation. By irradiating the line cod with 0.5 X 10s rad its storage life could be nearly doubled or extended by 10 days. This storage life extension is significant, and it should be pointed out that line cod control (unirradiated) had a 12 days1 storage life, whereas the commercial line cod fillets had 7 days' storage life. The difference in storage life may undoubtedly be attributed to environmental bacterial contamination of the fillets. From the bacteriological viewpoint it is interesting to note that the whole line cod reached the borderline of 1.0 X 107 bacteria per gram ât a much later time than the other test criteria (O-test, TMA and VAN) indicated. This is contrary to what was seen with the cod fillets and is probably caused by slower bacterial penetration into the whole fish. It should be kept in mind that all samples for bacterial testing of the whole cod were taken aseptically underneath the skin. The skin harbours much greater bacterial load than the flesh, and the metabolic products of the skin flora may diffuse into the flesh and therefore result in faster organoleptic changes (and chemical changes) than would be caused by the bacteria in the flesh itself. These results are therefore not comparable with those of Liston and Matches [10] as both skin and flesh were used for bacteriological testing in their study. Contrary to the irradiated .cod fillets (2.0 X 105 rad), the irradiated whole cod (0.5 X 105 rad) showed an increase in VAN which is similar to that found in unirradiated cod during storage. The TMA of whole cod behaved similarly to what was found in the study of the cod fillets. The O-tests were made on samples of skinned fillets cut from the whole cod and were evaluated in the same manner as the fillets. No anomaly was noted in the O-tests on whole cod. Re-irradiation of fish fillets from pre-irradiated whole fish has been reported by US workers [10, 26] in which industrial conditions of ir-radiation on board the fishing vessels and re-irradiation on shore were simulated. One experiment was conducted in this study by irradiating whole line cod

89 (pre-irradiation with 0. 5 X 105 rad), filleting after 3, 7 and 12 days' storage and re-irradiating the fillets with 1.5X 105 rad on the days of filleting. From Table XXI it may be seen that the post-irradiation storage life of the fillets cut from pre-irradiated whole cod appears to be inversely related to the storage time of the pre-irradiated whole cod. The longer the whole cod was stored the shorter was the post-irradiation storage life of the fillets. This agrees with Carver's results [26] . Further, it seems that the total storage life of re-irradiated cod fillets is only slightly longer than that of the whole, pre-irradiated cod. The total bacterial counts of the fillets reached the borderline of 1. 0 X lO7 per gram before the samples taken underneath the skin from the whole cod. When Table XXI is compared with Table XXII it may be seen that the commercial line cod fillets irradiated at the beginning of the experiment with 2.0 X 10s rad had a longer post-irradiation storage life but a slightly shorter total storage life than the re-irradiated cod fillets cut from pre-irradiated whole cod. The shortening of the post-irradiation storage life of the re-irradiated cod fillets is probably caused by inherent environmental contamination during filleting in the laboratory as suggested in Section 13. 3 and Table XXVII. The effect of environmental contamination was repeatedly noticed in the various experiments. This led to setting up an experiment where the effect of the sanitary care in handling during preparation (filleting, etc.) was evaluated. The results of this experiment are shown in Tables XXII and XXVII, where these factors appear very clearly. By using special care in sanitation in filleting in the laboratory, a storage life extension of 9 days was found beyond that of the commercial machine-cut cod fillets. Laboratory hand-cut and skinned fillets showed the shortest storage life of the three types which were all irradiated at a dose level of 2.0 X 105 rad. The importance of sanitation during preparation of fish products that are to be preserved by irradiation must be strongly emphasized. From the viewpoint of the Icelandic fishing industry it would appear feasible to irradiate whitefish (cod, haddock), lobster tails and shrimp in a shore-based irradiation centre on the southwest coast where the main whitefish grounds are located (see also Parts I and II). The irradiation centre could easily be provided with constant supply of very fresh raw material by using line-caught whitefish. The fishing vessels taking part in line fishing land their catch daily and the whole fish are of prime quality and of low bacterial load. This makes the advantages of the ship-board irradiation facilities of doubtful benefit for Icelandic conditions. In addition to irradiating ready-packed fillets the irradiation centre could also serve as a store-house for whole irradiated whitefish, which would be collected there and exported periodically. This would guarantee an even supply of market-fresh, whole whitefish preserved by radiation pasteurization.

15. SUMMARY AND CONCLUSIONS

The aim of this study was to investigate the effect of the raw material quality of cod resulting from three different methods of catching on the quality of the irradiated product and its effect on the storage life. In the irradiation studies both cod fillets (2.0 X 105 rad) and whole eviscerated cod

90 (0. 5 X 105 rad) were used. Some of the irradiated whole cod was filleted and the fillets re-irradiated (1.5 X 10s rad). The quality assessment tests used were sensory evaluation (O-test), trimethylamine (TMA), volatile acid number (VAN) and total bacterial counts (TBC) at 22°C. The O-test by a newly developed technique proved to be easy to carry out and gave reliable indication of the end-point of storage life. The TMA determination correlated fairly well with the O-test on cod irradiated at the dose levels used in this study. The VAN determination showed poor correlation with the O-test on cod irradiated above 1. 0 X 105 rad but fair for lower dose levels and the unirradiated samples. The borderline of total bacterial count was in this study placed at 1.0 X 107 per gram. Irradiated cod fillets reached that bacterial count at an earlier date than the O-test date of rejection indicated. However, bacterial counts of whole cod taken from underneath the skin reached the borderline count of 1.0 X 107 at a much later time than the end-point of storage life indicated by the O-test, TMA and VAN. The methods of catching which yield raw material suitable for radiation pasteurization of cod are cod caught by line and/or by trawl if landed daily. The net-caught cod was found to be unsuitable for this purpose. The commercial line cod fillets irradiated at a dose level of 2.0 X 105 rad had a storage life of 24 days at 0°-l°C. Whole line cod irradiated with 0.5 X 106 rad had a storage life of 21 days at 0°-l°C. Re-irradiated (1. 5 X 105 rad) fillets of the pre-irradiated (0. 5 X 105 rad) whole line cod had a shorter storage life than expected. Unirradiated commercial line cod fillets had a storage life of about 7 days at 0°-l°C. An experiment where the effect of good house keeping and sanitation was studied showed these factors to be of great importance, and line cod fillets handled with special care showed a storage life of 33 days at 0°-l°C. The conclusions reached from this study on radiation pasteurization of whole cod and cod fillets are as follows: As very fresh raw material, line-caught cod and haddock is always, available in Iceland, the radiation preservation should be performed in a shore radiation centre. This shore radiation centre could also process other seasonal seafoods (see Parts I and II). The dose level for irradiation of cod fillets is recommended to be of the order of 2. 0 X 105 rad or higher than suggested for the US domestic market because time and transport routes are longer. The filleting of cod should be done by machine to minimize bacterial contamination. Low-level radiation of whole, eviscerated cod was promising for export to markets preferring the whole fish to fillets.

91 ACKNOWLEDGEMENTS

The authors, G. Hannesson and B. Dagbjartsson of the Icelandic Fisheries Laboratories, wish to acknowledge the able assistance of the following people who have taken part in the project of radurization of lobster tails (scampi), deep sea shrimp and cod:

Dr. I. El-Rawi, IAEA Fellowship holder from Iraq Mr. C. Cannistraci, IAEA Fellowship holder from Argentina Miss G. Guevara, IAEA Fellowship holder from the Republic of the Philippines Mr. J.S. Colomer, IAEA Fellowship holder from Chile Mr. S.R. Agarwal, IAEA Fellowship holder from India Mr. R.H. Chen, IAEA Fellowship holder from China (Taiwan) Dr. A. Sorathesn, IAEA Fellowship holder from Thailand and Mr. H. Thorsteinsson, laboratory technician of the Icelandic Fisheries Laboratories, and other members of the staff of the Icelandic Fisheries Laboratories who so willingly and conscientiously performed their tasks as members of the taste panel.

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92 [15] MINISTRY OF HEALTH, Committee on Medical and Nutritional Aspects of Food Policy; Report of the Working Party on Irradiation of Food, H.M. Stationary Office, London (1964), [16] COLBY, J., SHEWAN, J.M., "The radiation preservation of fish", Fish as Food 4 (BORGSTROM, G., Ed.), Academic Press, New York (1965) 419. [17] DASSOW, J.A., MIYAUCHI, D., "Radiation preservation of fish and shellfish of the Northeast Pacific and Gulf of Mexico", Radiation Preservation of Foods, National Academy of Sciences Publication 1273 (1965) 53. [18] VON MEIR, G. W., "The economic feasibility of radiopasteurization of fish", The Technology of Fish Utilization: (2) Freezing and Irradiation of Fish ( KREUZER, R., Ed.), Fishing News (Books), London (1969) 504. [19] HOBBS, G., SHEWAN, J.M., "The present status of radiation preservation of fish and fishery products in Europe", The Technology of Fish Utilization: (2) Freezing and Irradiation of Fish (KREUZER, R., Ed. ), Fishing News (Books), London (1969) 488. [20] GORESLINE, H., "Status on legislation of irradiated foods", The Technology of Fish Utilization: (2) Freezing and Irradiation of Fish (KREUZER, R., Ed.), Fishing News (Books), London (1969) 478. [21] SKÚLADÓTTIR, U., Processing of Seafoods (Proc. Conf. in Icelandic with English summary), V.F.I. Reykjavik (1967) 13. [22] HANSEN, P. et al. , The Deep-sea Shrimp (Pandalus borealis), Biology-Fishery-Utilization, Fiskeri- ministeriets Forsôgslaboratorium, Copenhagen (1968). [23] HANSEN, P., AAGAARD, J. , "Freezing of shellfish", The Technology of Fish Utilization: (2) Freezing and Irradiation of Fish (KREUZER, R. , Ed.), Fishing News (Books), London (1969) 147. [24] DYER, W.J. , Report on trimethylamine in fish, J. Ass. off. agrie. Chem. 42 2 (1959). [25] AMPOLA, V. G. , CONNORS, J.T. , RONSIVALLI, L.J., Preservation of fresh, unfrozen fishery products by low level radiation, Fd Technol. , Champaign 23 (1969) 357. [26] CARVER, J.H., CONNORS, J.T., SLAVIN, J.W., "Irradiation of fish at sea", The Technology • of Fish Utilization: (2) Freezing and Irradiation of Fish (KREUZER, R., Ed.), Fishing News (Books), London (1969) 509. [27] AMPOLA, V.G., RONSIVALU, L. J. , Effect of preirradiation quality of eviscerated haddock on postirradiation shelflife of fillets, J. Fd.Sci. 34 (1969) 27. [28] LEARSON, R. I., RONSI VALU, L. J., Fishery Industrial Research 4, No. 7, US Department of the Interior, Fish and Wildlife Service, Bureau of Commercial Fisheries (1969) 249. [29] US ATOMIC ENERGY COMMISSION, Eighth Annual US Atomic Energy Commission Irradiation Contrac- tors' Meeting, USAEC, Washington, D.C. (1968) 298. [30] EUROPEAN INFORMATION CENTRE FOR FOOD IRRADIATION, Table III, Fd Irrad. 9 3 (1969). [31] CASTELL, C.H. et al., Grading for quality: 1. Trimethylamine values of fillets out from graded fish, J. Fish. Res. Bd Can. 15 (1959) 701. [32] HOLSTON, J., Fd Irrad. 9 3 (1969) 26.

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