135 Original paper Transactions of the Japan Society of Refrigerating and Air Conditioning Engineers, TransactionsReceived of the date Japan：April Society 14, of2018 Refrigerating；J-STAGE and Advance Air Conditioning published Engineers,date：May 31, 2018 Original Paper Vol.35,No.2(2018),pp.135-139,doi: 10.11322/tjsrae.18 -doi:10_OA 10.11322/tjsrae.18-10 _OA Received date: April 14, 2018; J-STAGE Advance publishied date: May 31, 2018

Biological Metabolism of Frozen Whale Meat at Subzero Temperatures in Relation to Thaw Rigor

Hideto FUKUSHIMA*† Naho NAKAZAWA** Koki YAMADA* Masahiro MATSUMIYA* Ritsuko WADA*** Toshimichi MAEDA***

* Department of Marine Science and Resources, Nihon University (1866 Kameino, Fujisawa, Kanagawa 252-0880) ** Department of Food Science and Technology, Tokyo University of Marine Science and Technology (4-5-7, Konan, Minato-ku, Tokyo 108-8477) ***Department of Food Science and Technology, National Fisheries University (2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595)

Summary In many cases, frozen whale meat in the Japanese market is prepared before rigor mortis (pre-rigor). A serious problem for frozen whale meat is the occurrence of thaw rigor, which is the strong development of rigor mortis during thawing. To prepare frozen whale meat without thaw rigor and maintain a high meat pH, the temporal changes in adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) contents of frozen meat stored at -2.5, -5.0, -7.5, and -10°C were investigated. The rate of decrease of ATP was higher than that of NAD at all storage temperatures. ATP nearly disappeared after holding the meat at -2.5°C for a few days; however, NAD existed yet, so pH decreased thereafter. ATP levels were maintained for a long period at a temperature of -5.0 to -10°C, resulting in the occurrence of thaw rigor. Compared to the muscles of fish such as tuna, the rates of decrease of ATP and NAD were extremely slow in whale meat.

Keywords: ATP, Thawing drip, Frozen meat, Mink whale, NAD, pH, subzero temperature

1. Introduction maintain freshness because of recent developments in refrigerating equipment8). ATP, the energy source for The development of postmortem rigor in mammals, muscle contraction, inhibits the thermal denaturation such as cows and pigs, is markedly slower than that of proteins11-12). Furthermore, ATP inhibits the in fish1-2). Therefore, the muscle tissue of domestic denaturation of myofibrillar protein and autoxidation mammals intended for consumption is subjected to a of oxymyoglobin to metmyoglobin during frozen comparatively long aging process for 2 weeks to 1 storage13-14). Therefore, fresh frozen fish meat month. During the aging process, postmortem rigor containing highly concentrated ATP shows high dissolves, taste components gradually increase, and freezing tolerance, but requires careful treatment to muscle becomes edible meat1). In contrast, because prevent thaw rigor during thawing. the whale is a marine mammal, its processing greatly We studied freezing characteristics focusing on differs from that of domestic mammals. Whale meat highly fresh frozen bigeye tuna15). We found that in the Japanese market is mainly a residual product nicotinamide adenine dinucleotide (NAD), a from resource or biological surveys. The body is glycolytic coenzyme, decreased during storage at dissected and the meat block is frozen immediately -10°C. Additionally, the anaerobic glycolytic after the survey. Therefore, frozen whale meat in the pathway could be inhibited during and after thawing. market is highly fresh and in a pre-rigor state. The Interestingly, NAD in bigeye tuna meat decreased frozen meat quickly undergoes thaw rigor, shrinking during storage at -10°C, while ATP showed a minimal the meat, and dripping during thawing, greatly decrease at the same temperature. Thus, it was decreasing the meat quality3-4). This has also been possible to prepare thawed meat at neutral pH by reported for fresh frozen fish, such as skipjack5), decreasing NAD at -10°C and then gradually thawing sardine6), and bigeye tuna7). The membrane structure to 0°C. Fish meat at neutral pH shows high water of the endomysium and sarcoplasmic reticulum is holding property and sensory evaluation in destroyed as ice crystals are generated upon freezing8). comparison to that at low pH15). Thaw rigor is caused by leakage of Ca2+ from the In this study, we focused on highly fresh frozen sarcoplasmic reticulum, rapid consumption of whale meat. Whale meat contains high protein and adenosine triphosphate (ATP), and contraction of the low lipid contents as well as free amino acids, such as muscle during thawing9-10). balenine, and thus is an important marine food The thaw rigor occurs likely because it is possible resource16). However, frozen whale meat exhibits to quickly freeze various biological samples to high thawing drip containing taste components, as

†E-mail:fukushima.hideto＠nihon-u.ac.jp

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described above. Therefore, changes in the biological 12°C until the central temperature reached 5.0°C. It characteristics of whale meat at subzero temperatures took about 10 minutes from -2.5 °C and 25 minutes were investigated. The results showed that storage at from -10 °C for thawing up to 5°C. Thawing drip (%) subzero temperature not only decreases ATP, but also was calculated as the difference in weight of meat NAD, as has been observed in frozen bigeye tuna before and after thawing. meat.

3. Results 2. Experimental 3.1 Change in ATP content 2.1 Materials Frozen whale meat blocks of approximately 150 g The ordinary muscle tissue of the minke whale stored at -50°C were transferred to a freezer at -2.5, Balaenoptera acutorostrata caught in the North -5.0, -7.5 and -10°C and preservation experiments Atlantic Ocean was used in this study. The frozen were conducted. The changes in ATP content during mass of 15 kg of highly fresh whale meat was storage at four subzero temperatures are shown in Fig. provided by Kyodo Senpaku Co., Ltd. (Tokyo, Japan). 1. The initial amount of ATP was 11.9-15.5 µmol/g. The frozen mass was divided into blocks of After storage at -2.5°C, ATP content rapidly approximately 150 g (25 x 50 x 125 mm) by decreased to 0.47 µmol/g after 2 days and nearly Marukou-shouji Inc. (Yamaguchi, Japan). Meat disappeared to 0.11 µmol/g after 5 days. In contrast, blocks were stored at -50°C in the lab until use. At the ATP content after storage at -5.0°C remained at 4.86 beginning of the experiment, the blocks were µmol/g even after 21 days. Following storage at -7.5 transferred to a freezer at -2.5, -5.0, -7.5, and -10°C. and -10°C, ATP contents were 1.61 and 1.92 µmol/g after 108 and 133 days, the final day of storage, 2.2 ATP content respectively. According to the method described in our previous report17), ATP contents of frozen meats at subzero temperatures were analyzed. Frozen meat samples about 5 g were quickly removed from meat block in the freezer room at -5.0°C. The meat sample was also quickly homogenized in 15 mL of cold 10% perchloric acid, and then centrifuged at 1,500 x g for 3 min. The supernatant was filtrated through filter paper of No. 2 and the pH was adjusted to neutral. The ATP content was determined by high- performance liquid chromatography.

2.3 NAD content NAD content was measured from the same extract obtained as described in Section 2.2 according to the method of Ehira et al.18). Fig. 1 Changes in the amount of ATP in frozen minke 2.4 pH measurement whale meat at storage temperatures of -2.5, -5.0, -7.5, The pH of whale meat at subzero temperatures and and -10°C. immediately after thawing was measured according to the method of Bito et al.6). Frozen meat samples 3.2 Change in NAD content about 5 g were quickly removed from meat block in The changes in NAD content during storage at four the freezer room at -5.0 °C. Thawed meat samples subzero temperatures are shown in Fig.2. The initial about 5 g were also removed immediately after amount of NAD was 0.59 µmol/g. The rate of thawing up to 5.0 °C. And then, meat sample was also decrease of NAD was greatest under storage of -2.5°C, quickly homogenized in 25 mL of cold 20 mM but NAD content was 0.34 µmol/g 5 days later. NAD iodoacetic acid. pH was measured with pH meter content was 0.35 µmol/g at -5.0°C after 21 days, 0.25 (Horiba, F-52). µmol/g at -7.5°C after 108 days, and 0.27 µmol/g at - 10°C after 133 days. The temperature dependency 2.5 Thawing drip was similar to that of ATP, although the rate of Frozen block meat about 150 g was packed into a decrease was slower than that of ATP at the same polyethylene bag without air under normal pressure temperatures. and thawed under running water at approximately

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Fig. 2 Changes in the amount of NAD in frozen minke whale meat at storage temperatures of -2.5, -5.0, -7.5, Fig. 3 Changes in pH of frozen and quickly thawed and -10°C. minke whale meat at storage temperatures of -2.5, - 5.0, -7.5, and -10°C. (a); Changes in pH before and 3.3 Change in pH after thawing after storage at -2.5 and -5.0°C. (b); The changes in pH before and after thawing during Changes in pH before and after thawing after storage storage at different temperatures are shown in Fig. 3. at -7.5 and -10°C. The initial pH of the frozen whale meat was 6.7–6.9, which is nearly neutral. The pH of the frozen meat 3.4 Changes in thawing drip after storage at -2.5°C rapidly decreased to below 6.0 The changes in thawing drips at the four storage after 2 days, even at subzero temperatures. temperatures are shown in Fig. 4. Initially, a large Furthermore, the pH decreased to 5.7 after 5 days, and amount of ATP and NAD remained and thawing drip pH after thawing was nearly the same. After storage was approximately 30 %. After storage at -2.5°C, at -5.0°C, the pH decreased more gradually than at - thawing drip decreased to approximately 3 % and 2.5°C, and was around 6.0 after 21 days. At that time, thaw rigor was not observed. At other storage the pH did not change before and after thawing. temperatures, a large amount of thawing drip Similarly, pH gradually decreased during storage at - (approximately 20 %) and thaw rigor were observed 7.5 and -10°C, but did not decrease to below 6.0 after after the final storage—21 days at -5°C storage, 108 108 and 133 days, respectively. At these two storage days at -7.5°C, and 133 days at -10°C. These results temperatures, the changes in pH before and after suggest that frozen whale meat containing 1.5–2.0 thawing were very large. ATP and NAD were also µmol/g ATP exhibits thaw rigor and high drip during present at the end of storage days as shown in Fig. 1 thawing. and 2, and thus the pH under these conditions would decrease over additional storage time.

Fig. 4 Changes in thawing drip (%) of quickly thawed minke whale meat after storage at -2.5, -5.0, -7.5, and

-10°C.

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Next, changes in thawing drip for 30 days of Normally, lactic acid may have been generated storage at -2.5°C were evaluated (Fig. 5). As shown with the resolution and reproduction of ATP, in Fig. 4, thawing drip rapidly decreased to below 5 % decreasing pH9). Based on the changes in pH (Fig. 3), after 3 days. During storage, thawing drip gradually lactic acid may have been produced during storage increased and reached approximately 10 % after 30 even at subzero temperatures. The rate of decrease of days. ATP was clearly greater than that of NAD in frozen whale meat, in contrast to in frozen fish meat. However, it was recently reported that the rate of decrease of ATP in chub mackerel Scomber japonicus frozen meat was greater than that of NAD19). Thus, the relationship between the rates of decrease of ATP and NAD is not the same in all fish species. Interestingly, the pH of thawed meat after the storage period at -5.0, -7.5 and -10°C did not decrease to the minimum, 5.7 as same as that at -2.5°C (Fig. 3). These meats occurred thaw rigor, so it was possible that various enzymes and substrates were expressed as thawing drip and the pH of meat did not decrease so much. The changes in thawing drip (Fig. 4) were well-

Fig. 5 Changes in thawing drip (%) of quickly thawed correlated with ATP content, and thaw rigor occurred minke whale meat after storage at -2.5°C for 30 days in the presence of ATP. ATP contents were 1.5–2.0 µmol/g after the storage period at -7.5°C and -10°C, (n = 3). but both samples showed thaw rigor and expressed

more than 20 % thawing drip. Therefore, it is 4. Discussions important to reduce ATP content to below 1.0 µmol/g to inhibit thaw rigor. Changes in thawing drip after In this study, changes in biochemical properties at storage at -2.5°C were observed during the 30 days subzero temperatures effective for inhibiting thaw 4) (Fig. 5). The thawing drip decreased to below 5 % rigor in frozen whale meat were investigated. The after 3 days, and then gradually increased to 10 % by rate of decrease in ATP content in frozen whale meat the final storage day. This may be because protein was clearly slower than that in fish, such as bigeye 5-7, 15) was no longer preserved by ATP, resulting in protein tuna . In the case of frozen bigeye tuna meat, 20) 7) denaturation during freezing to increase drip . In ATP nearly disappeared at -5.0°C within 10 hours . addition, low pH would relate the denaturation and In contrast, ATP content of frozen whale meat at increase of thawing drip. -5.0 °C remained about initial half after 2 weeks In this study, the ATP content of highly fresh (Fig.1). ATP in the muscle is thought to be resolved frozen whale meat decreased during storage at -2.5°C by various enzymes, such as myofibrillar ATPase. At and thus, thaw rigor was inhibited. In contrast, the the same time, ATP is reproduced through the rates of decrease of NAD were slower than those of anaerobic glycolytic pathway, and so on. Therefore, ATP at four storage temperatures, -2.5, -5.0, -7.5, and the result in Fig.1 showed that the decreasing rate of -10°C. Unfortunately, it is difficult to maintain a ATP exceeded the reproducing rate. neutral pH in whale meat because NAD content does Similarly, changes in NAD during the storage at not decrease more rapidly than ATP content, as in four subzero temperatures were investigated (Fig. 2). bigeye tuna. On the other hand, it has been The initial concentration of NAD was 0.6 µmol/g, reconfirmed that thaw rigor is inhibited by the storage which was lower than that of bigeye tuna at 0.9 15) at -2.5 °C for a few days. However, it was noted that µmol/g . Additionally, the rate of decrease in NAD the decrease of pH at -2.5 °C is fast, resulting in differed. In bigeye tuna, NAD content decreased to protein denaturation and the increase of thawing drip 0.2 µmol/g at -10°C after 2 days and 0.1 µmol/g after in the later storage. 5 days. In contrast, the NAD content of frozen whale meat decreased to 0.42 µmol/g at -2.5°C after 2 days and 0.34 µmol/g after 5 days. If NAD content in bigeye tuna meat is below 0.2 µmol/g, the anaerobic Acknowledgement glycolytic pathway does not function during thawing and the pH of meat would no longer decrease15). In We thank Prof. late Yutaka Fukuda for drafting this frozen whale meat in this study, ATP nearly study. In addition, we thank Mr. Hitoshi Funahashi disappeared after 5 days at -2.5°C. At that time, the and Mr. Mitsuhito Ogihara from Kyodo Senpaku Co., NAD content remained at 0.34 µmol/g, so it was Ltd. for providing valuable samples. possibly not to stop the anaerobic glycolytic pathway. 4

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