RAINBOW TROUT {Salmo irideus) PRODUCED IN FINLAND VI. Free amino acid composition of refrigerated and frozen rainbow trout Ritva-Liisa Sihvola, Jorma J. Laine & Fritz P. Niinivaara University of Helsinki, Institute of Meat Technology Received November 21, 1967 Fish is considered a good source of protein and the amino acid composition is the deter- mining factor when evaluating the nutritive value of protein (Konosu et al. 1956). During the second year of cultivation the Finnish rainbow trout has a protein content of above 17 per cent and the seasonal variations in the protein content are around 2 per cent (Laine et ai. 1967). When stored in ice at + 4 •• + 6°C the changes in the total amino acid content during storage seem to be influenced more by the differences between in- dividual fish than by time (Niinivaara et ai. 1966). The purpose of this work was to follow the changes of free amino acids in rainbow trout (Salmo irideus) produced in Finland during storage in ice at -f- 4 •• + 6°C and at 18°C. Material and methods The trout weighed approximately 250 grams each and were transported to the laboratory alive. After killing and bleeding they were gutted. For refrigerated storage the fish were kept in ice at +4 •• -f- 6°C on perforated trays and new ice was substituted daily. The fish were packed in polyethylene bags and frozen at 30 °C and stored at 18°C. Free amino acids were determined with an amino acid analyzer (Technicon Auto- Analyzer). Samples of the refrigerated fish were taken after 24 and 72 hours and 10 days. Samples of the frozen fish were taken after 3, 8 and 13 months. The control sample was prepared from fresh gutted rainbow trout. Samples for the determination of free amino acids were prepared as follows: The fish were homogenized in a blender. An amount of 10 grams of the homogenate was weighed and 90 ml of absolute alcohol was added. The sample was then homogenized with an Ultra-Turrax homogenizer. After homogenization the sample was filtrated and washed with 80 per cent alcohol (Welcher 1963). 50 The sample was then evaporated in a vertical vacuum evaporator and double distilled water was added until all the alcohol was evaporated. The pH of the sample was then adjusted to 2.87. Results The results obtained from the fish stored in ice at -f- 4 ■ • -f- 6°C are presented in Table 1. They indicate that the total amount of free amino acids decreased during storage. The development in the individual amino acids followed the same pattern but correlation with the control could not be shown in many instances. Freezing did not affect the amounts of free amino acids as much as refrigeration (Table 2). During storage at — 18°C, the total amount of free amino acids decreased, however, and the results regarding individual acids followed the same pattern as the results ofrefrigerated fish compared with the control. Discussion The studies performed indicated a decrease in the amounts of free amino acids during refrigerated and frozen storage of rainbow trout (Tables 1 and 2). Changes were more distinct in the refrigerated than in the frozen fish. Quantitative results indicated that the amounts of threonine, serine, alanine, iso-leucine, leucine, valine, ornithine, and lycine at the beginning of refrigerated and frozen storage increased at first slightly compared with the control, decreasing later during the storage. The same phenomenon was more distinct with glutamic acid and proline. The amounts of aspartic acid, methionine, tyrosine, phenylalanine, y-aminobutyric acid, and arginine remained almost stable throughout the experiments. A steady decrease occurred in the amounts of glycine, histidine, and, in connection with refrigeration, more distinctly also in tryptophane. These three components formed 76.2 per cent of the total amount offree amino acids in the control sample and constituted the main reason for the decrease of total amounts of free amino acids in rainbow trout during refri- gerated and frozen storage. With the technique used, the taurine, urea, 3-hydroxyproline, glucosamine, norvaline, [3-alanine, ethanolamine, hydroxylysine, and a-amino-n-caproic acid could not be quanti- tatively determined. The results of these substances were presented proportionally according to the analytical values. Summary Changes in the amounts offree amino acids in rainbow trout during refrigerated storage in ice at -f- 4 •• + 6°C and in frozen storage at 18°C were investigated. The control sample was prepared directly from the killed, bleeded, and gutted fish. The results indicated that the total amounts of free aimino acids decreased in the re- frigerated and in the frozen fish. These decreases depended mostly upon the steady decrease of the three quantitatively largest components, namely histidine, tryptophane, and glycine. Histidine formed 36.1 per cent, tryptophane 25.0 per cent, and glycine 15.1 per cent of the Table 1. Free amino acid content of gutted trout stored in ice. Control 24 hours 72 hours 10 days taurine mg/g ** * * % urea mg/g *** * ** ** /o 3-hydroxyproline mg/g * * * * o/ ./o asparticacid mg/g * * 0.008 0.004 % 0.565 0.312 threonine mg/g 0.045 0.079 0.052 0.035 % 2.436 5.223 3.675 2.726 serine mg/g 0.023 0.070 0.050 0.028 % 1.245 4.630 3.534 2.181 glutamic acid mg/g 0.031 0.154 0.182 0.106 % 1.678 10.185 12.862 8.255 proline mg/g 0.058 0.115 0.058 0.046 % 3.140 7.606 4.099 3.583 glysine mg/g 0.279 0.255 0.135 0.233 % 15.106 16.865 9.541 18.146 alanine mg/g 0.087 0.101 0.080 0.104 % 4.710 6.680 5.654 8.100 glucosamine mg/g * * 0/ . valine mg/g 0.029 0.055 0.037 0.033 % 1.570 3.638 2.615 2.570 methionine mg/g 0.015 » 0.015 0.013 % 0.812 1.060 1.012 norvaline mg/g * * * % iso-leucine mg/g 0.016 0.026 0.021 0.018 % 0.866 1.720 1.484 1.402 leucine mg/g 0.030 0.052 0.039 0.030 % 1.624 3.439 2.756 2.336 tyrosine mg/g 0.013 � 0.022 0.016 % 0.704 1.555 1.246 phenylalanine mg/g 0.023 0.033 0.030 0.074 % 1.245 2.183 2.120 5.763 [3-alanine mg/g * * * o//o ethanolamine mg/g * * * % y-amino-butyric acid mg/g 0.016 * * % 0.866 hydroxy-lysine mg/g * * % ornithine mg/g 0.010 0.035 0.013 0.009 % 0.541 2.315 0.919 0.701 lycine mg/g 0.037 0.061 0.150 0.275 % 2.003 4.034 10.601 21.417 tryptophan mg/g 0.461 * 0.023 * % 24.959 1.625 histidine mg/g 0.666 0.476 0.487 0.252 % 36.058 31.481 34.417 19.626 a-amino-n-caproic acid mg/g * o//o arginine mg/g 0.008 * 0.013 0.008 % 0.433 0.919 0.623 mg/g 1.847 1.512 1.415 1.284 % 99.996 99.999 100.001 99.999 *analytical value < 5; ** analytical value 5 —10; ***analytical value > 10. Table 2. Free amino acid content of gutted trout stored at 18°C. Control 3 months 8 months 13 months taurine mg/g ** * ** * 0/la urea mg/g *** *** *** •»* 0//o 3-hydroxyproline mg/g * * o//o asparticacid mg/g * 0.004 * 0.005 % 0.218 0.359 threonine mg/g 0.045 0.074 0.025 0.081 % 2.436 4.037 1.983 5.823 serine mg/g 0.023 0.034 0.022 0.060 % 1.245 1.855 1.745 4.313 glutamic acid mg/g 0.031 0.165 0.086 0.035 % 1.678 9.002 6.820 2.516 proline mg/g 0.058 0.097 0.086 % 3.140 5.292 6.183 glycine mg/g 0.279 0.149 0.124 0.077 % 15.106 8.129 9.833 5.536 alanine mg/g 0.087 0.109 0.080 0.094 % 4.710 5.946 6.344 6.758 glucosamine mg/g * * % - - - - valine mg/g 0.029 0.042 0.028 0.055 % 1.570 2.291 2.220 3.954 methionine mg/g 0.015 0.012 0.015 0.009 % 0.812 0.655 1.190 0.647 norvaline mg/g * * o//o iso-leucine mg/g 0.016 0.022 0.012 0.020 % 0.866 1.200 0.952 1.438 leucine mg/g 0.030 0.035 0.029 0.055 % 1.624 1.909 2.300 3.954 tyrosine mg/g 0.013 0.013 0.016 0.011 % 0.704 0.709 1.269 0.791 phenylalanine mg/g 0.023 0.030 0.007 0.023 % 1.245 1.637 0.555 1.653 (3-alanine mg/g * * • 0//o ethanolamine mg/g * * ai y-amino-butyric acid mg/g 0.016 0.009 0.011 % 0.866 0.491 0.872 hydroxy-lysine mg/g * o/ _ ornithine mg/g 0.010 0.013 * * % 0.541 0.709 lycine mg/g 0.037 0.038 0.163 0.033 % 2.003 2.073 12.926 2.372 tryptophan mg/g 0.461 0.468 0.630 0.228 % 24.959 25.532 49.960 16.391 histidine mg/g 0.666 0.519 0.013 0.519 % 36.058 28.314 1.031 37.311 a-amino-n-caproic acid mg/g * * ai arginine mg/g 0.008 * * * % 0.433 mg/g 1.847 1.833 1.261 1.391 % 99.996 99.999 100.000 99.999 'analytical value < 5; *‘analytical value 5—10; ‘“analytical value > 10. 53 total amount of free amino acids in the control sample. In connection with many other amino acids a decrease in their amounts was noted during storage. Changes appearing in refrigerated fish were more distinct than in frozen fish. Recognition and appreciation is extended to the Institute of Dairy Science, University of Helsinki, for cooperation and for making the amino acid analyzer available for this study. REFERENCES Konosu, S., Katori, S., Ota, R., Eguchi, S. & Mori, T. 1956. Amino acid composition of fish muscle protein. Bull. Jap. Soc. Sci. Fish. 21: 1163. Laine, J. J., Varesmaa, Elina & Niinivaara, F. P. 1967. Rainbow trout (Salmo irideus) produced in Finland. 111. Seasonal variations in rainbow trout. J. Sci. Agric. Soc. Finland 39; 133 Niinivaara, F. P., Sihvola, Ritva-Liisa & Laine, J. J. 1966. Rainbow trout (Salmo irideus) produced in Finland. I. Bacterial spoilage and amino acid composition of fresh rainbow trout during refrigerated storage. Ibid. 38: 210. Welcher, F. J. 1963. Standard methods of chemical analysis. Voi. 2. Industrial and natural products and noninstrumental methods, Part A. D. Van Nostard Company, Inc., New York. SELOSTUS TUTKIMUKSIA SUOMESSA KASVATETUSTA KIRJOLOHESTA ( Saima irideus) IV. Kirjolohen vapaissa aminohapoissa tapahtuvat muutokset jäähdytyksen ja pakastuksen aikana Ritva-Liisa Sihvola, Jorma J. Laine & Fritz P. Niinivaara Helsingin Tliopisto, Lihateknologian laitos Suoritetussa tutkimuksessa seurattiin kirjolohen vapaissa aminohapoissa tapahtuvia muutoksia säily- tyksen aikana jäähileessä + 4 ••• -f 6°C:ssa ja pakastettuna 18°C:ssa. Vertailu suoritettiin kontrollinäyt- teeseen, joka valmistettiin tuoreesta, peratusta kirjolohesta. Tulokset osoittivat, että vapaiden aminohappojen määrät alenivat säilytyksen aikana sekä jäähileeseen pakatuissa että pakastetuissa kaloissa. Aleneminen johtui lähinnä kolmen kvantitatiivisesti suurimman aineen määrissä tapahtuneesta vähenemisestä. Histidiinin osuus oli 36.1 %, tryptofaanin 25.0 % ja glysiinin 15.1 % kontrollinäytteen vapaiden aminohappojen kokonaismäärästä. Muidenkin aminohappojen koh- dalla oli havaittavissa laskua varastoinnin aikana. Muutokset pakastetunkalan kohdalla olivat pienemmät kuin jäähileeseen pakatussa kalassa.