RAINBOW TROUT {SALMO IRIDEUS) PRODUCED IN FINLAND I. Bacterial spoilage and amino acid composition of fresh rainbow trout during refrigerated storage Fritz P. Niinivaara, Ritva-Liisa Sihvola and Jorma J. Laine University of Helsinki, Institute of Meat Technology Received October 19, 1966 The first problem with the decay of foodstuffs under natural conditions is usually microbiological, while enzymatic and ohemical deterioration occurs after the spoilage by micro-organisms has started (4). The development of micro-organ- isms during the storage of fresh fish is accompanied by decomposition of the muscle carbohydrates, proteins, and lipids; and because of variations in composition of the muscle of different species and the complex nature of the bacterial populations in- volved, no consistent degradative pattern can be expected (8). On the other hand, the chemical and physical composition of an individual fish depends on the season, sex, age, food, and environment (2, 7). Psychrophilic bacteria occurring commonly in water, air and soil form the natural microflora on the external surfaces of fish, while the flesh and internal organs of healthy freshly caught fish are considered to be bacteriologically sterile (8). The microbial invasion into the originally sterile flesh of the fish begins from the surface slime through the skin, but invasion also takes place through the gills and viscera (7). In spoiling fish, defects in color, odor and taste are usually the most striking changes but also questions in connection with public health must be taken seriously into consideration because clostridia, including Cl. botulinum, Cl. telani and Cl. sporogenes occur in the intestines of fish (7, 9). The storage life of trout produced on Danish trout farms is about nine days in ice, for whole and for gutted trout (1). Vacuum packing under the same con- ditions clearly improved the keeping quality (3). Irradiation of vacuum packed gutted trout prolonged the storage life further (5). Fish is generally considered a rather good source of animal protein. The value of protein does not depend merely upon the total amount of proteins but also on the amino acid composition (9). https://www.c-info.fi/en/info/?token=zRmIAQrmR-98dn5R.0Kurw_HKUWlsQoK2E_q1hQ.ZZDNprwcqc6PbtgpZWLrp3tCDLEl1iboto208NO2ngWcNZPdklGng9HGOnBBl42-hq1xgDPFGeSaIii_WtixsxCqHX3fuXvYPeDmoJGNdLk5niFr3cqEebX3ZD4_Dq4NNHPApyaNiq2YP_gSs81d6tDghIXw3rFi68KTAH_F5MtOijPr50X-iFKvf3JKoSn-CLy-APif9dnfPygF4lyQ8raEZiXxJ6m3pg 211 In this sense, essential amino acids are the determing factor when the quality of fish is judged on this basis. In some cases the amounts of essential amino acids are almost the same even in different species of fish (6). In the present study the bacteriological spoilage, organoleptic quality and amino acid composition of rainbow trout (Salmo irideus) produced in Finland was investigated. Material and methods Experiments were carried out with 2-year old trout cultivated in Sysmä. The mean weight of the fish was 242 grams (range 162—302 gram). The control fish were transported to the laboratory alive, the rest of the fish was first killed and gutted. All the fish was in the laboratory within 4 hours. In approximate analyses the whole fish contained 70.4 % water, 8.5 % fat, 17.7 % protein, 3.6 % ash, its pH was 6.70. The corresponding figures for the gutted fish were 71.1 %, 8.0 %, 17.6 %, 3.2 % and pH 6.40, respectively. The samples were kept 1) in air, 2) in ice, 3) in polyethylene bags and 4) in vacuum bags at +4 1-6° C throughout the experiment. Bacteriological exp eriments. For each sample 11 grams of fish were aseptically weighed in 99 ml of 0.9 % NaCl-solution. The sample was then homogenized and the necessary decimal dilution series was prepared. Experiments were carried out with living fish, after storage periods of 4 and 30 hours, and 5, 7 and 13 days. All samples were tested for total viable aerobic counts on SPC-agar (Orion), for total coliforms on VRB-agar (Orion), and the vacuum-packed samples also for anaerobic sulphide producers on iron-sulphite agar (Orion). Changes in the pH in all samples were measured during storage. Incubation for total viable aerobes was 72 hours at 20° C, for coliforms 48 hours at 37° C and for anaerobic sulphide producers 120 hours at 37° C. Organoleptic evaluation. Organoleptic evaluation was made with raw fish and after it had been cooked in fysiological NaCl-solution for 10 minutes at 90° C. The evaluation panel consisted of four tasters who gave scores as follows: Appearance (scores from 0 to 4) Structure ( » »0 to 4) Color ( » »0 to 4) Odor ( » »0 to 2) Flavor ( » »0 to 6) maximum score 20 Besides these scoring numbers, faults were also noted verbally. Determination of amino acids. Amino acid determinations were carried out with an amino acid analyzer (Technicon Auto-Analyzer). Determinations were made on living fish, after 4 hours, and on fish stored in ice for 3, 6 and 9 days. Samples were prepared as follows: The fish was homogenized in a blender. An amount of 3 grams of the homogenate was weighed out in 1000 ml of HCI (20 %). 212 The hydrolysis was carried out in a vertical condenser for 20 hours at 110° C. Then the sample was evaporated in a vertical vacuum evaporator. Thereafter the sample was washed until the pH remained between 2 and 3. After filtration the pH was adjusted to 2.87 and the sample was diluted into 1000 ml of double distilled water and a portion of 1 ml was used for the analyses. Results Bacteriological experiments. The total viable aerobic counts of different samples are presented in Fig. 1. The results show clearly that counts are highest in the samples kept in air. After five days of storage when the relative differences between the samples were at their greatest, the counts in air were 445 X 106/gram, in polyethylene bags 47 X 106/gram, in vacuum bags 19.51 x 106 / gram and in ice 84 X 103/gram. In ice the amount of bacteria increased slowly and after 7 days of storage it was 210 X 103/gram. The bacterial invasion in the ice-stored fish was first clearly noted after 10 days of storage. The amount of total coliforms was considered as an indication of hygiene. Fig. 2 reveals that the tendency in the different samples followed the same pattern as that for the total bacterial counts. Counts were highest in the fish kept in air, while storing in ice was the most effective method also against coliforms. The Fig. 1. The total viable aerobic counts in gutted trout on SPC-agar at +4 +6° C. 213 relative differences between the different types of treatments in this test (Fig. 2), however, were smaller than in the previous test (Fig. 1). Vacuum-packed trout was also tested for anaerobic sulphide producers in iron-sulphide agar. The amount of this type of organisms was unexpectedly high (Fig. 3). Fig. 4 shows the changes in the pH during storage. The pH initially decreased naturally in all the samples, but thereafter an increase did not take place until after 12 days. It occurred first in fish kept in air, then in ice, while it was slowest in the fish packed in polyethylene bags and vacuum bags. On the other hand, in the two latter instances the pH reached its lowest level, being after 10—13 days of storage 5.85—5.90. Fig. 2. The total counts of coliforms in gutted trout on VRB-agar at +4 -f 6° C. Fig. 3. The anaerobic sulphide-producing bacteria in gutted trout stored in ice on iron-sulphide agar at +4 1-6° C. 214 Organoleptic evalution. Table 1 shows the organoleptic quality of living and gutted (4 hours) fish. These results served as controls for the different types of packages (Tables 2,3, 4 and 5) during storage. In comparing the organoleptic quality of the different types of packages (Tables 2,3, 4 and 5), differences were clearly observed. In air the fish was unacceptable after 6 days (Table 2), while in ice this happened after 11 days (Table 3) and in Table 1. Organoleptic quality of fresh and gutted (4 hours) trout. Appear- Struc- Color Odor Flavor Total ance ture score Fresh Raw 3.5 4 3.5 2 pale red neutral Cooked 4-442 4.5 18-i- -turbid salmon neutral led 4 hours Raw 3+ 4- 3+ 2 pale soft light neutral Cooked 4-4-4 2 4.5 18 turbid soft salmon red Fig. 4. The pH of gutted trout during storage at +4 +6° C. 215 Table 2, Organoleptic quality of gutted trout stored in air. Time Appear- Struc- Color Odor Flavor Total days ance ture score 2 Raw 3 4 3+2 dry rigor light Cooked 4 4 4 2 4 18 rancid 3 Raw 3 3.5 3+ 2 dry post light rigor Cooked 4 3.5 4 2 3 16.5 soft rancid dry 5 Raw 13 3 1 slimy soft color- rancid turbid less Cooked 3 2.5 4 1 1.5 12 turbid soft rancid spoiled 6 Raw 0.5 1 1 1- slimy loose milky rancid Cooked 0.5 0 1 1-0 2+ slimy loose milky rancid completely juicyless spoiled Table 3. Organoleptic quality of gutted trout stored in ice. Time Appear- Struc- Color Odor Flavor Total days ance ture score 2 Raw 3 4 3 2 turbid light Cooked 3 3.5 3 2 3 14.5 turbid dry pale dry 5 Raw 2.5 3 3 2 turbid dry pale Cooked 2.5 3.5 3 2- 2.5 13+ turbid dry pale odorless dry rancid 8 Raw 13 11 turbid soft colorless odorless Cooked 3.5 2 0.5 0.5 1 7.5 turbid dry colorless odorless rancid denatured 11 Raw 0 10 0 slimy dry soft colorless spoiled Cooked 2 2 0 0 0 4 slimy soft colorless spoiled rancid denatured 216 Table 4. Organoleptic quality of gutted trout stored in polyethylene bags. Time Appear- Struc- Color Odor Flavor Total days ance ture score 2 Raw 4 4 3 2 rigor pale Cooked 4 4 3-2 4,5 17 + pale 5 Raw 4 3 3-2 soft pale Cooked 4 3 3.5 2- 4 16+ juicy soft pale off-odor rancid 11 Raw 2.5 1 2- 0.5 slimy soft pale rancid Cooked 3 3 2- 1.5 2.5 12- turbid soft pale rancid 13 Raw 2 0 10 slimy soft colorless spoiled Cooked 2 0 0 0 1 3 turbid dis- colorless sour rancid integrated Table 5. Organoleptic quality of gutted trout stored in vacuum bags. Time Appear- Struc- Color Odor Flavor Total days ance ture score 2 Raw 4 3 4 2 soft red Cooked 4 4-425 19- juicy 5 Raw 4 3.5 3 2 firm pale Cooked 4 4 3 2 4+ 17 + pale off- flavor 11 Raw 2 3 2 1 slimy soft pale off- odor Cooked 3 2 2.5 1 3 11.5 dis- pale off- off- integrated odor flavor 13 Raw 2 2.5 2 1 slimy soft pale off- odor Cooked 2.5 2 1.5 1 3 10 turbid dis- color- off- sour integrated less odor 217 polyethylene bags after 13 days. In vacuum bags the fish was considered edible still after 13 days of storage. Determination of amino acids. Experiments made with the fish stored in ice (Table 6) reveal that no great changes in the total amino acid composition occurred during the experiment. The relative changes between the different amino acids were rather similar in the tests, and the quantitative changes probably depended upon the individual fish investigated. Glutamic acid was the greatest component (15.713 —17.217 %) followed by lysine (12.38—14.353 %). With the method used, 17 different amino acids could be detected in amounts from a Table 6. Amino acid content of gutted trout stored in ice. Control 4 hours 3 days 6 days 9 days aspartic acid mg/g 8.24 10.108 8.06 7.226 9.310 % 9.67 9.138 8.72 9.136 9.650 threonine mg/g 2.93 4.641 4.04 3.372 4.283 % 3.44 4.196 4.37 4.263 4.440 serine mg/g 4.65 4.375 3.53 3.185 3.850 % 5.46 3.955 3.82 4.027 3.990 glutamic acid mg/g 14.02 17.380 15.34 13.585 16.610 % 16.46 15.713 16.61 17.175 17.217 proline mg/g 1.265 0.117 % 1.144 0.121 glycine mg/g 5.22 6.475 5.20 3.675 7.327 % 6.12 5.854 5.63 4.165 7.595 alenine mg/g 4.92 7.446 5.36 4.628 5.727 % 5.77 6.732 5.80 5.851 5.936 valine mg/g 4.09 5.226 5.03 4.251 4.913 % 4.80 4.725 5.44 5.374 5.093 cystein mg/g 1.408 1.35 1.239 % 1.273 1.46 1.566 methionine mg/g 3.37 3.526 2.83 2.781 2.930 % 3.95 3.188 3.06 3.516 3.037 iso-leucine mg/g 4.67 4.279 4.19 3 406 4.017 % 5.48 3.869 4.53 4.306 4.164 leucine mg/g 6.50 8.340 7.03 6.375 6.943 % 7.63 7.540 7.61 8.060 7.197 tyrosine mg/g 3.31 3.017 3.31 3.017 3.380 % 3.88 2.728 3.58 3.814 3.504 phenylalanine mg/g 3.57 3.630 4.01 3.630 3.740 % 4.19 3.282 4.34 4.589 3.877 lysine mg/g 10.55 15.799 11.71 10.065 13.847 % 12.38 14.283 12.68 12.725 14.353 histadine mg/g 3.50 3.920 6.65 1.960 3.290 % 4.10 3.544 7.20 2.478 3.410 argenine mg/g 5.62 9.776 4.71 6.611 6.190 % 6.59 8.838 5.10 8.358 6.416 mg,g 85.16 110.611 92.35 79.006 96.474 % 99.92 1C0.002 99.95 99.403 100.000 218 few mg/g to about 10 mg/g. However, the amounts of proline and cystein did not exceed these figures in all instances. Proline was present in all samples, but cystein could not be detected in the control nor in the 9-day sample. Discussion The keeping quality of rainbow trout varied considerably according to the packing method used in an experiment under laboratory conditions. In many instances bacteriological and organoleptical results were not directly correlated with each other. Fish stored in air spoiled most rapidly both bacteriologically and organoleptically. Storage in ice was most effective from the bacteriological standpoint (Figs. 1 and 2). When the experiments were carried out at +4 1-6° C there may have been a somewhat lower temperature in fish stored in ice compared to the other packing methods used. On the other hand, bacteria could be washed out from the surface of the fish when the ice was changed; not until after 7 days of storage did the total viable count exceed 105 bacteria/g. The bacterial counts in fish packed in poly- ethylene and vacuum bags were similar throughout the experiment, although the total viable count and the coliform count in vacuum were somewhat lower (Figs. 1 and 2). The high count of anaerobic sulphide producers must, however, be taken seriously under consideration in connection with vacuum-packed fish (Fig. 3). These could be detected immediately after gutting in amounts which exceeded the total viable aerobic count. Because of the relatively high anaerobic counts, vacuum-packing cannot be recommended at present as a method for fresh trout. In this respect, more information is needed about the pathogenity of these bacteria. The changes in pH (Fig. 4) showed a typical initial decrease. An increase in pH occurred first with the fish kept in air. In polyethylene and vacuum bags the pH reached its lowest level, namely 5.85—5.90, after 10 and 13 days of storage. Only thereafter was there a slow increase in the pH. Organoleptic studies showed that vacuum-packed trout was still edible after 13 days (Table 5). Slime-formation occurred only after 10 days, when degradation in structure, color and odor began to become more distinct. Fish packed in polyethyl- ene bags held their shelf life for 11 days but after that complete spoilage set in rapidly (Table 4). Fish in ice showed unfavorable features of sensory quality most rapidly. Already after 2 days the flesh was lacking in juice and tasted dry (Table 3). The washing effect of melted ice may account for this. Comparing these results with those obtained in the Danish experiments (1, 2, 3 and 5) it can be noted that storage in ice showed similarresults in the organoleptical tests (2). Vacuum packing had a favourable effect in organoleptic quality in both cases, but the bacterial counts were lower in Denmark (3). However, the Danish experiments were carried out at lower temperatures. The temperature, however, is not the only factor involved, because big differences have been noted depending on handling, season and sex (2). Radiation pasteurization in connection with vacuum packing (5) seems to improve the keeping quality of fresh trout considerably, but 219 the public health aspects should be examined in detail before vacuum packing can be recommended as a method for storing fresh trout. Total amino acid analyses showed that 17 different amino acids were detectable with the method and concentration used (Table 6). There were no considerable changes during storage between the different amino acids. Quantitative differences are thought to depend on the variations between individual fish. In the next study an investigation will be made on the volatile amino acid composition of fresh trout during storage. Summary Bacteriological spoilage, organoleptical quality and amino acid composition of fresh trout were studied during storage at +4 (-6° C. Experiments were carried out with living fish (control), with fish 4 hours after killing and during storage. The fish were kept in air, in ice and packed in poly- ethylene and vacuum bags. It was observed that the type ofpacking considerably influences both the bacte- riological and organoleptical quality. These changes were not, however, directly correlated with each other. In connection with vacuum packing, the amounts of anaerobic sulphide producing bacteria were so high that this aspect needs a detailed investigation before vacuum packing can be recommended for fresh trout. The amino acid composition of iced trout changed only slightly during storage. Current experiments concerning changes in volatile amino acid contents will provide additional information in this respect. Recognition and appreciation is extended to the Institute of Dairy Science, University of Helsinki, for cooperation and for making available the amino acid analyzer in this study. REFERENCES (1) Hansen, P. 1963. Fat oxidation and storage life of iced trout. I. Influence of cutting. J. Sci. Fd Agric. 14; 781. (2) Hansen, P. 1964. Fat oxidation and storage life of iced trout. 11, The influence of sex and season. Ibid. 15: 344. (3) Hansen, P. & Jorgensen, B. V. 1965, Storage life of vacuum-packed iced trout. I. Influence of packing matenal. Ibid. 16: 150. (4) Ingram, M. 1962. Microbiology, biochemistry and food. Recent Advances in Food Science, Vol. 2: 307. London, (5) Jorgensen, B. V. & Hansen, P, 1966. Storage life of vacuum-packed iced trout. 11, Influence of radiation pasteurization. J. Sci Fd Agric. 17: 140, (6) 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. (7) Shewan, J. M. 1961. The microbiology of sea-water fish. In Fish as Food, Voi 1: 487. New York. (8) Tarr, H. L. A. 1954. Microbiological deterioration of fish post mortem, its detection and control. Bact. Reviews 18: 1. (9) Venkataraman, R. & Chari, S. T. 1957. Amino acid composition of some marine fishes. Ind. Jour. Med. Res. 45: 77, 3 220 SELOSTUS; TUTKIMUKSIA SUOMESSA KASVATETUSTA KIRJOLOHESTA (SALMO IRIDEUS) I. Tuoreen kirjolohen säilyvyys ja aminohappokoostumus Fritz P. Niinivaara, Ritva-Liisa Sihvola & Jorma J. Laine Helsingin Yliopisto, Lihateknologian laitos Tuoreen peratun kirjolohen bakteriologista pilaantumista, organoleptista laatua ja aminohappo- koostumusta seurattiin koesarjalla, joka suoritettiin -f 4 -f 6° C:ssa. Kokeita tehtiin elävästä kalasta, 4 tuntia teurastuksen ja perkauksen jälkeen sekä säilytyksen aikana. Kaloja säilytettiin kokeen aikana perkaamattomina sellaisenaan, jäähileessä, muovikalvoon pakattuna ja vakuumipakkauksessa. Havaittiin, että pakkaustavalla oli selvä vaikutus kalan bakteriologiseen ja organoleptiseen laa- tuun. Muutokset eivät kuitenkaan olleet suorassa korrelaatiossa keskenään. Suoritetun tutkimuksen valossa ei vakuumipakkausta voida suositella tuoreen kirjolohen pakkaustavaksi ennen kuin klostriidi- kysymys on perusteellisesti selvitetty. Anaerobisten sulfidinmuodostajien määrä oli odottamattoman korkea vakuumipakatuissa kaloissa. Aminohappokoostumuksessa esiintyi sangen vähäisiä vaihteluita. Liukoisten aminohappojen tutkiminen tulee selventämään kokonaiskuvaa tuoreen kirjolohen aminohappojen kohdalla tapahtu- vista muutoksista säilytyksen aikana.