Fish Sci (2011) 77:289 DOI 10.1007/s12562-011-0346-7 OBITUARY In Memoriam: Professor Michizo Suyama (1923–2011) Ó The Japanese Society of Fisheries Science 2011 Professor Michizo Suyama, an Honorary Member of the Japanese Society of Fisheries Science and Former Professor of Tokyo University of Fisheries, passed away on January 19, 2011 He was 87 years old Professor Suyama was born in April 1923 in Tokyo He graduated from the Imperial Fisheries Institute in 1943 and became a Research Associate at the Imperial Fisheries Institute in 1945 In 1949, the National School Establishment Law created the Tokyo University of Fisheries by incorporating the Imperial Fisheries Institute and the Faculty of Fisheries This new institute was placed under the jurisdiction of the Ministry of Agriculture and Forestry Professor Suyama remained at the new Tokyo University of Fisheries, becoming an Associate Professor in 1960 and a Full Professor in 1974 He retired from the university in 1987 The focus of Professor Suyama’s research activities was marine food chemistry His various fields of research and educational interest included studies on the amino acid composition of fish protein, extractive components, taste-active components, and smells of volatile substances His contributions to these fields and those on the improvement and modification of an amino acid analyzer are excellent and have contributed to developments in fisheries science Professor Suyama also contributed to the Japanese Society of Fisheries Science, serving first as a member of the Board of Directors, then as Vice President and finally President He received the Japanese Society of Fisheries Science Award of Merit in the fields of nitrogenous extractive components from aquatic animals in 1987 His social contributions as a scientist were invaluable In 1999, he received The Order of the Sacred Treasure, Gold Rays with Neck Ribbon Professor Suyama educated and inspired many young people with his profound knowledge and warm personality He was especially fanatical in his desire to improve the taste and palatability of food We offer heartfelt our condolences to the family of Professor Suyama Takaaki Shirai Associate Professor, Tokyo University of Marine Science and Technology 123 Fish Sci (2011) 77:291–299 DOI 10.1007/s12562-011-0326-y ORIGINAL ARTICLE Fisheries Growth and fatness of 1975–2002 year classes of Japanese sardine in the Pacific waters around northern Japan Atsushi Kawabata • Hirotsune Yamaguchi Seigo Kubota • Masayasu Nakagami • Received: March 2010 / Accepted: 23 December 2010 / Published online: 26 February 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract We examined individual growth and fatness in the 1975–2002 year classes of Japanese sardine Samples were collected at the feeding grounds in the Pacific waters off northern Japan during drastic fluctuations in the population in the 1970s to 2000s Growth rates for ages 1–3 of the 1979–1988 year classes, which included low-recruitment year classes subsisting during the high population levels of the 1980s, were apparently slower than for other year classes There was no obvious trend when comparing year classes, growth during the first year of life (age 0), and maximum body length (BL) at age C5 The condition factors (CF, indicating fatness) for adult sardines of BL C19 cm in the 1979–1983 year classes during the maximum population level of the mid-1980s were significantly lower than for other year classes However, there were no apparent trends in CF variations for small sardines of BL \19 cm The apparent decreases in growth rate and fatness were strongly related to the cumulative sum of population abundance that each year class experienced It is thought that insufficient food owing to the density-dependent effect of an abundant population at feeding grounds resulted in a A Kawabata (&) National Research Institute of Fisheries Science, Fisheries Research Agency, Japan, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan e-mail: abata@affrc.go.jp H Yamaguchi Japan Fisheries Information Service Center, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan S Kubota Á M Nakagami Tohoku National Fisheries Research Institute, Fisheries Research Agency, Japan, 25-259 Shimomekurakubo, Samemachi, Hachinohe, Aomori 031-0841, Japan decrease in the growth rate for small-bodied sardines that are investing their energy intake in body growth, and a decrease in fatness for large-bodied adults that are accumulating fat for the next reproduction Keywords Age Á Body length Á Condition factor Á Density effect Á Growth Á Population Á Sardinops melanostictus Introduction Japanese sardines Sardinops melanostictus spawn in the coastal waters along the south of Japan, near Kuroshio, in winter–spring, and migrate northward in summer–autumn to forage in the Pacific waters [1–4] The population biomass has fluctuated drastically in the past, in synchronization with global climate change, and this fluctuation has impacted Japanese fisheries and Japanese society [5] The Japanese sardine population in the Pacific waters increased in the 1970s due to the occurrence of comparatively high recruitment levels in 1973, 1977 and 1978 They became very abundant in the 1980s due to very high sequential recruitment from 1980 to 1987 (Fig 1) These population increases were thought to be due to favorable environmental conditions for reproduction and an increase in the number of spawned eggs The increased number of eggs was due to the accumulation of spawners from several generations due to the comparatively long life span of this species In the 1980s, over one million tons of sardine were fished, mainly by purse seiner, in the feeding grounds off Sanriku and the eastern Hokkaido regions of northern Japan However, the population declined through the 1990s, reaching a very low level in the 2000s The sardine catch in the Pacific waters off northern Japan decreased to only 0–4000 tons in the 2000s 123 292 Fish Sci (2011) 77:291–299 Abundance (x10 ind.) 600 Catch (x10 t) 1.6 500 400 300 1.2 Materials and methods 0.8 From 1975 to 2005, we collected samples of Japanese sardine during the fishing season from commercial landings at the Hachinohe Port, an important fishing port in northern Japan (Fig 2) These were caught by purse seiner off the Sanriku coast The fishing season off the Sanriku coast varied with sardine population abundance, extending to 10 months (from late April to early February) during the 1980s due to the high population levels at that time, but shortening to only a few summer months in recent years due to low population levels Vessel survey samples taken by drift net or pelagic trawl were also collected off the Pacific coast of northern Japan in May–January from 1998 to 2003 in order to supplement the decreasing commercial catch sample (Fig 2) We measured scaled body length [BL (cm)] and determined age in years (t) by the method of counting annual rings on the scales [16] for a total of 12,161 specimens These specimens were assigned to the 1975–2002 year classes We measured body weight [BW (g)] and gonad weight [GW (g)] for a subset of 11,918 specimens Internal abdominal fat [FW (g)] was also weighed for a subset of 7,633 specimens The condition factor (=coefficient of fatness, CF) was calculated from the obtained biometric data using the equation CF = 103 (BW – GW)/BL3 Fat weight index was also calculated (=105 FW/BL3) Having examined the correlation between BL and CF and also the seasonal variation of CF, as described later, we used the mean CF for separate BL ranges of samples during May– November (BL \19 cm) or May–December (BL C19 cm) to examine the effect of density To estimate the BL at the start of each age, the von Bertalanffy growth curve was fitted by the method of least squares to the age and BL data of each year class: Lt = L? [1 - exp(-K (t - t0))], where Lt is the BL at the start of age t, L? is the asymptotic BL, K is the rate at which BL tends toward the asymptote, and t0 is the age when BL = The beginning of each age, the birthday, was set to 1st March, as this was midway through the main spawning season The age data used to fit the curve was t ? d/365, where d is the number of days from 1st March to the sampling date In fitting the curve, L? was limited to less than 30 cm, which is the length of the largest sardine that has been caught [17] For the catch of Japanese sardines in the Pacific waters off northern Japan, we referred to the fishery statistics collected by the Fisheries Research Agency and to previous reports [18–20], and used them as an index of abundance at 200 0.4 100 1970 on variations in body length, growth rate and fatness among year classes 1975 1980 1985 1990 1995 2000 2005 Year Fig Annual total population abundance (all ages, available after 1977, gray and unfilled bars) and recruitment (age 0, gray bars) of Japanese sardine in Pacific waters (after Wada and Jacobson [6], Nishida et al [7]), and catch in the Pacific off the Sanriku and the eastern Hokkaido areas of northern Japan (closed diamonds and solid line) It is known that the individual growth rates of Japanese sardine in the coastal waters off eastern Hokkaido decreased in the 1980s compared with the 1970s [8–10] A similar phenomenon was observed at the past population peak in the 1930s [11, 12] This slow growth was considered to be subject to a density-dependent effect of the abundant population [8–10] However, in these studies, sampling was restricted to the coastal waters of eastern Hokkaido and so they did not examine differences in growth rates among year classes There has not been enough discussion about whether the growth rate in a year class is affected by its own year class recruitment abundance or rather by the population abundance (standing stock of all ages) in Japanese Pacific waters The growth rate of sardines recovered after the 1990s, but recent changes in growth related to population abundance have not yet been examined in depth Variations in fatness relating to both growth and population abundance have also not been examined in detail in previous studies It was reported that fatness in the 1980s year classes was lower than in the 1970s year classes when comparing same-age fish [10] However, there is a positive correlation between fatness and body length, as described later in this study The decrease in the fatness of same-age fish in the 1980s probably reflected shorter body lengths, rather than fatness per se The effect of fish density on fatness should be examined clearly by comparing fish of the same body size In this study, we examine the growth and fatness of 1975–2002 sardine year classes in relation to the drastic fluctuations in the population abundance of sardines in the Japanese Pacific waters, based on biometric data collected over 31 years We also discuss the density-dependent effects of recruitment abundance or population abundance 123 Fish Sci (2011) 77:291–299 °N 45 °N 50 Oyashio Hokkaido 40 Kuroshio Extension Pacific Ocean Spawning ground Sanriku 40 Hachinohe Port Fig Location of the sampling area, and schematic view of the oceanographic current systems and the spawning ground of the Japanese sardine population in Pacific waters (after Watanabe [13], Kondo [2], Kuroda [14], Kobayashi and Kuroda [15]) Cross and diamond symbols indicate the locations of purse seiner sampling (n = 340) and survey vessel sampling (n = 126), respectively 293 35 140 the feeding grounds For the total population abundance (standing stock of all ages) and the recruitment of each year class (cohort abundance at age 0), we referred to the estimates gained from the virtual population analysis performed in Wada and Jacobson [6] and Nishida et al [7] We examined variations in both the Lt at each age and the CF for different BLs with respect to population abundance and year-class recruitment among the 1975–2002 year classes To examine the effect of population abundance on the growth of a year class, we used the cumulative sum of population abundance for the period from age to the previous age (t - 1) as an index of the abundance that the P year class born in year x experienced: xþtÀ1 Ni ; where Ni i¼x is the total population abundance in year i Population abundance estimates were available for years after 1977 (Fig 1), so year classes after 1977 were examined Results Growth The BLs and ages of the specimens ranged from 6.4 to 24.4 cm and from to years, respectively Figure shows age and BL observations and the fitted von Bertalanffy growth curves for typical year classes The number of observations for each year class depended on the ease of sampling (i.e., year-class abundance) and ranged from 53 (1990 year class) to 2,332 (1986 year class) 130 145 140 150 30 160°E 150°E Figure shows the estimated BL of each year class at the start of each age from to 9, as estimated from the fitted growth curves Gaps are due to a lack of observations for some ages in some year classes The maximum BL at age or older was about 21–22 cm for all year classes, although there was a lack of observations for some year classes Estimated BL at age for each year class was 13–16 cm Small BLs (13–14 cm) at age occurred in high-recruitment (more than 150 billion individuals, as occurred in 1980–1987) year classes during high population levels, and in low-recruitment (less than 50 billion individuals, as occurred in 1979, 1998, 1999 and 2001) year classes during low population levels (Fig 1) Also, large BLs (15–16 cm) at age occurred in both the comparatively high recruitment (94 billion individuals) year class of 1978 and the low-recruitment year classes in the 1990s–2000s There was no obvious relation between estimated BL at age and either recruitment of year classes or total population abundance Estimated BLs at ages 2–4 differed distinctly between year classes Estimated BLs of the 1979–1987 year classes were 15–16 cm at age 2, 17–18 cm at age 3, and 18–19 cm at age (Fig 4) These BLs at ages 2–4 were smaller than those of the 1975–78 year classes by 1–2 cm and smaller than those of 1989–2002 year classes by 2–3 cm Table shows the estimated BLs, the growth rates at age t (increase in BL (Lt?1 - Lt)/Lt) and the fitted von Bertalanffy equation parameters for typical year classes living during different population abundance levels Growth rates 123 294 Fig Relationships between age and body length as well as fitted von Bertalanffy growth curves for typical year classes Fish Sci (2011) 77:291–299 Body length (cm) 25 20 15 10 1979 n = 169 n = 1,728 1987 1989 n = 559 25 20 15 10 1982 n = 210 n = 271 1988 1995 n = 54 0 10 10 10 Observation Growth curve during age in the 1979 and 1982 year classes (i.e., for high population levels of more than 300 billion individuals) were apparently slower than those for the 1987–1989 year classes (i.e., when the population was decreasing) and for the 1995 year class (i.e., for low population levels of less than 30 billion individuals) (Fig 1) However, the estimated BLs at age for the 1979 and 1982 year classes were not very different to those for other year classes The growth rates during the early ages for the year classes corresponding to high population levels (e.g., 1979 and 1982) were slower than those for year classes corresponding to decreasing or low population levels, as indicated by the comparatively small K parameters Furthermore, the estimated BL at age for the 1988 year class was smaller than those for the 1975–1978 and 1989–2002 year classes (Fig 4; Table 1) The growth rate during age of the 1988 year class should have been slow, as it is for the 1979–1987 year classes, although there was a lack of age-1 BL observations However, the estimated BLs at ages and of the 1988 year class were similar to those for the 1975–1978 and 1989–2002 year classes (Fig 4; Table 1) The BL of the 1988 year class improved with age The 1987 year class showed a similar improvement in BL with age (Fig 4; Table 1) Recruitment in these slow-growing year classes was not high Recruitment was high (more than 150 billion individuals) in the 1980–1987 year classes, but was low (less than 50 billion individuals) in the 1979 and 1988 year classes (Fig 1; Table 1) Significant correlations were found between the recruitment and the estimated BLs at ages 2–4 for the year classes (P \ 0.01) However, there were some outliers, such as the 1979 year class (Fig 5) The 1979 year class subsisted during the high-level populations of the 1980s On the other hand, sardine populations in the Japanese Pacific waters were at very high levels during 1980–1989 123 Age Body length: L t (cm) 24 Age t 22 20 18 16 14 12 1975 1980 1985 1990 1995 2000 Year class Fig Estimated body length at the beginning of each age for the 1975–2002 year classes (306–554 billion individuals) The 1979–1986 year classes were at ages 1–3 at this time, and had small BLs at ages 2–4 (Figs 1, 4) Figure shows the relationships between the estimated BL of each year class at ages 1–4 and the cumulative sum of the population abundance (as explained in ‘‘Materials and methods’’) in Japanese Pacific waters Significant negative correlations were noted between the BL of each age group from to and the cumulative sum of population abundance, though the BL at age was not significantly correlated This suggests that the total population abundance affected the growth rates during ages 1–3 To examine the effects of the recruitment and total population abundance on the growth of sardines, we conducted multiple regression analysis for the estimated BL at ages 2–4 for the year classes against recruitment and the cumulative sum of population abundance (Table 2) For BLs at all ages from to 4, the population abundance exhibited a large absolute value for the partial regression coefficient and was a significant explanatory variable, whereas the recruitment was not a significant variable It Fish Sci (2011) 77:291–299 295 Table Estimated body length (Lt, cm) at the beginning of each age (t), increase in body length [(Lt?1 - Lt)/Lt, %], and the von Bertalanffy equation parameters L?, K and t0 for typical year classes Year class L1 1979 13.7 13.7% 15.6 1982 14.1 12.1% 1987 14.0 17.3% 1988 ND 1989 1995 L2 L3 L4 L5 L6 L? K t0 10.7% 17.3 8.5% 18.7 6.9% 20.0 5.7% 21.2 30.0 0.12 -3.98 15.8 9.3% 17.3 7.3% 18.5 5.8% 19.6 4.7% 20.5 26.1 0.15 -4.06 16.5 10.3% 18.1 6.5% 19.3 4.2% 20.1 2.8% 20.7 22.0 0.36 -1.81 ND 16.6 13.8% 18.9 6.5% 20.1 3.2% 20.7 1.7% 21.1 21.5 0.63 -0.34 15.5 16.7% 18.0 7.9% 19.5 4.0% 20.3 2.1% 20.7 ND ND 21.2 0.59 -1.20 15.1 25.9% 18.9 8.1% 20.5 3.0% 21.1 1.1% 21.3 ND ND 21.5 0.93 -0.30 Body length (cm): Lt Body length (cm): Lt 17 t=1 16 n = 20 r = -0.524 15 t=1 t=2 19 16 n = 27 r = -0.824** 18 15 12 21 17 14 16 13 1979 15 12 14 22 20 n = 26 r = -0.829** 18 16 1979 13 2001 t=2 19 n = 20 r = -0.468 15 17 1988 14 20 17 20 14 t=3 t=4 n = 28 r = -0.838** n = 25 r = -0.851** 21 200 400 600 400 800 1200 22 21 20 19 t=3 t=4 n = 26 r = -0.953** n = 25 r = -0.940** 21 19 20 20 18 17 18 19 1979 16 100 200 18 300 19 17 1979 100 200 300 Recruitment (109 ind.) Fig Relationships between the estimated body length (Lt) at the beginning of each age (t) and recruitment for the 1975–2002 year classes (**P \ 0.01) was the total population abundance rather than the recruitment that strongly affected the BLs of sardines at ages 2–4 Fatness Almost all specimens, including adults near the maximum BL, had small gonads Most specimens had large amounts of subcutaneous and internal abdominal fat Fat weight index was positively correlated to the CF (r = 0.560, n = 7,633, P \ 0.01) The CFs of all specimens were in the range 7.9–17.2, with an average of 12.6 There was a weak but significant trend for sardines with a larger BL to have a higher CF (r = 0.395, n = 11,918, P \ 0.01) A comparison of the CF at the same age between year classes could reflect the differences in BL mentioned above 16 18 500 1000 1500 500 1000 1500 2000 Cumulative sum of population abundance (10 ind.) Fig Relationships between the estimated body length (Lt) at the beginning of each age (t) for the 1977–2002 year classes and the cumulative sum of population abundance for the period from age to age (t - 1) (**P \ 0.01) Therefore, the variation in CF between year classes was examined for separate BL ranges Also, to examine seasonal variations in CF, monthly mean CFs for each year class was determined The means for all year classes were then averaged (Fig 7) The mean CF in each month can be seen to decrease in winter: December–February for BLs \19 cm or in January for BLs C19 cm The foraging season for Japanese sardine in the Pacific waters is summer and autumn [21] To examine the variations in CF among year classes, we used the comparatively high CF values obtained from samples during May–November for BL \19 cm or during May–December for BL C19 cm as the values that were considered to occur in the foraging season The mean CFs by BL range in each year class are shown in Fig The mean CFs for BL C19 cm were about 13–14 123 296 Fish Sci (2011) 77:291–299 Table Results from a multiple regression analysis of the estimated body length (Lt) at the beginning of each age from to against the recruitment (1011 ind.) of each year class and the cumulative sum of population abundance for the period from age to age (t - 1) (1011 ind.) Dependent variable Explanatory variable L2 (n = 26) Recruitment Partial regression coefficient 0.0362 Standard error Standard partial regression coefficient T value P value 0.354 0.024 0.10 0.920 Population abundance -0.352 0.089 -0.933 3.98 0.001** L3 (n = 26) Recruitment Population abundance 0.0311 -0.219 0.218 0.037 0.023 -0.975 0.14 5.96 0.888 0.000** L4 (n = 23) Recruitment -0.111 0.180 -0.104 0.62 0.545 Population abundance -0.117 0.023 -0.847 5.01 0.000** for most of the year classes, but were significantly lower (11.7–12.4) for the 1979–1983 year classes The mean CFs of each year class for BL = 17–19 cm and BL = 15–17 cm were about 12–13 and about 11–12, respectively, and those for BL \15 cm ranged from 10 to 12 There were no apparent trends in CF variation for BL\19 cm, which was similar to what was seen for BL C19 cm The mean CFs of large-bodied sardines with BL C19 cm during each year are shown along with the catch in Fig The CF values in 1983–1988 were in the range 11.4–12.2, lower than in most other years During this time, population levels in the Japanese Pacific waters were high (Fig 1), and the catch levels around northern Japan (an index of abundance in the feeding grounds) were at a maximum There was a significant negative correlation between CF and the catch (r = 0.578, n = 26, P \ 0.01) These large-bodied sardines of low CF mainly consisted of 1979–1983 year classes The high abundance in the feeding grounds must have affected the fatness of large-bodied sardines Discussion We compare our results with growth patterns for other small pelagic fishes that show biomass fluctuations like Japanese sardine Previous studies have reported densitydependent growth for some small pelagic fishes The western North Pacific chub mackerel (Scomber japonicus) undergoes a seasonal migration similar to that of Japanese sardine Their body lengths at age C1 are dependent on their growth in age and are negatively correlated with the year-class strength [22] The body size (length, mass) at age of the Pacific herring (Clupea pallasii) from the southwest coast of Vancouver Island is negatively related to parental biomass, owing to a pre-recruit effect at age [23] The body length at age of Pacific Hokkaido spring spawning herring is highly dependent on growth during age 0, and exhibited weak density-dependent effects of yearclass strength [24] Thus, the body sizes at older ages in these species are determined by their growth early in life, 123 Mean CF 14 BL >19 cm 13 17-19 cm 12 15-17 cm 19 cm 13 17-19 cm 15-17 cm 19 cm Catch in numbers SD Fig Annual changes in mean CF of BL C19 cm sardines and catch in the Pacific waters off Sanriku and the eastern Hokkaido areas of northern Japan (after Murakami and Kobayashi [18], Wada [10], Yamaguchi and Kawabata [20], fisheries statistics of FRA) year classes (1979–1983) It seems that small-bodied sardines were more affected by the density than largerbodied sardines The mean BL of sardines caught during the 1980s in the waters off eastern Hokkaido (further from the spawning ground) was greater than that of sardines caught in the coastal waters off Sanriku [20] This may mean that larger-bodied sardines can forage farther afield than smaller sardines and so reduce the density effect 123 298 According to previous studies based on analyses of ancient documents relating to Japanese regional fisheries, extra big catch years due to explosive increases in the Japanese sardine population in Pacific waters have occurred only five or six times since the sixteenth century [3, 11, 28] These previous periods of population increase seemed to last no longer than a few decades The recent explosive population increase in the 1980s was considered to have lasted for about a decade Only year classes (1980–1987) had outbreaks contributing to the explosive increase in population during the 1980s (Fig 1) This explosive population increase (outbreak) caused an obvious density effect, as mentioned above Changes in biological characteristics owing to the density effect, such as an apparent decrease in growth rate or fatness, were conspicuous and seemed to be unusual for Japanese sardine Additionally, the condition of spawning sardines (i.e., accumulation of body fat during the previous summer’s feeding migration) affects their gonadal development and the quality and quantity of eggs [29–31] Kawasaki and Omori [32] state that the density effect at the feeding ground in the 1980s caused the condition of the spawners and the egg quality at the spawning ground to deteriorate The outbreak was not necessarily a favorable situation for the sardine population During the long intervals between outbreaks of the population, the year classes that occur show relatively low recruitment and biological characteristics that might be regarded as normal The outbreak of Japanese sardines was a phase variation that coincided with global climate change [33] This does not indicate a recovery of the sardine population; instead it may be a short-term, disadvantageous state for the population Acknowledgments We would like to thank the editor and two anonymous reviewers for their constructive comments and suggestions We also thank Ms J Momosawa, Ms N Kubo, Mr M Kawamura and other staff at the Tohoku National Fisheries Research Institute for their assistance and support in this study This study was funded by the Program of Marine Fisheries Stock Assessment and Evaluation for Japanese Waters from the Fisheries Agency of Japan This paper is a contribution to the Study for the Prediction and Control of Population Outbreak in Marine Life in Relation to Environmental Change (POMAL) of the Agriculture, Forestry and Fisheries Research Council (AFFRC) References Kondo K (1980) The recovery of the Japanese sardine—the biological basis of stock-size fluctuations Rapp P-v Re´un Cons Int Explor Mer 177:332–354 Kondo K (1988) General trends of neritic-pelagic fish populations—a study of the relationships between 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Fish Sci (2011) 77:431–437 DOI 10.1007/s12562-011-0336-9 ORIGINAL ARTICLE Food science and technology Accumulation of gamma-oryzanol in teleost Reiko Nagasaka • Takamitsu Kazama Hideki Ushio • Hiroshi Sakamoto • Kenichi Sakamoto • Shuichi Satoh • Received: 24 December 2010 / Accepted: 14 February 2011 / Published online: March 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Japanese people consume crop bran, which contains relatively high amounts of gamma-oryzanol (ORZ), as foodstuffs and food materials We have recently confirmed that ORZ inhibits NF-jB activation, activates peroxisome proliferator-activated receptor gamma (PPARc), and increases plasma adiponectin levels ORZ is therefore expected to improve lipid and carbohydrate metabolisms and various inflammatory diseases, i.e., metabolic syndrome including type diabetes ORZ administration also allowed fish, such as rainbow trout, yellowtail, and red sea bream, to accumulate protein through enhancement of lipid and carbohydrate metabolisms In the present study, we have investigated ORZ accumulation levels in mouse and rainbow trout administered ORZ-containing feed Although mouse muscle and liver hardly contained ORZ, muscle tissues of every fish species accumulated higher amounts of ORZ These findings suggest that rainbow R Nagasaka Á T Kazama Á H Ushio Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 5-7 Konan 4, Minato, Tokyo 108-8477, Japan H Sakamoto Á K Sakamoto Sakamoto Feeds Co Ltd., 3-216-1 Matsugishi-cho, Choshi, Chiba 288-0836, Japan S Satoh Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 5-7 Konan 4, Minato, Tokyo 108-8477, Japan Present Address: H Ushio (&) Laboratory of Marine Biochemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan e-mail: aushio@mail.ecc.u-tokyo.ac.jp trout, red sea bream, and yellowtail accumulate ORZ in muscle Keywords Accumulation Á Gamma-oryzanol Á Hydroxycinnamic acid derivatives Á Physiological function Introduction Hydroxycinnamic acid derivatives (HADs), observed ubiquitously in plants, have some physiological functions It is reported that curcumin, a major active ingredient of turmeric in curry, and caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, are inhibitors of nuclear factor (NF)-jB activation [1, 2] and that they exhibit antioxidant activity [3, 4] On the other hand, one of the major bioactive HADs in rice bran oil, gamma-oryzanol (ORZ), is a mixture of ferulic acid esters of triterpene alcohols and phytosterols [5] Because the bran of cereals, including rice, is produced worldwide and the resources of these compounds are potentially enormous, effective utilization of the compounds has been highly desired and investigated ORZ from rice bran has been suggested to possess the capability of lowering serum cholesterol levels [6, 7], to offer anti-inflammatory [8] and anti-cancer effects [9], and to function as an antioxidant [10] trans-Ferulic acid, which results when rice bran oil is produced, also has an antioxidant effect [11, 12] We have recently demonstrated that ORZ suppresses NF-jB activation, consequently inhibiting inflammatory responses of macrophage cell line RAW264.7 [13] and improving inflammatory bowel disease [14] The transcription factor NF-jB is widely expressed in many tissues and plays important roles in immune systems [15] It has been reported that NF-jB activation is also associated with 123 432 insulin resistance [16, 17] Adiponectin expressed in and secreted from adipose tissue has been postulated to play an important role in the modulation of glucose and lipid metabolisms in insulin-sensitive tissues such as liver and skeletal muscle Plasma adiponectin levels are decreased in the obese and insulin-resistant state [18, 19] We have indeed demonstrated that ORZ enhances adiponectin secretion by the inhibition of NF-jB activation [20] Our recent papers have also demonstrated that ORZ prevents ethanol-induced liver injury [21] and attenuates mast cell degranulation [22] ORZ is thus expected to improve lipid and carbohydrate metabolisms, imbalanced inflammatory responses, and the related diseases, i.e., metabolic syndrome including type diabetes [23] Since it is difficult for us to ingest adequate cereal bran continuously, the development of other ORZ sources is important for our continuous ingestion of ORZ We here show that some fish species accumulate ORZ at high levels compared to mammals and that ORZ-accumulating culture fish might be useful for the novel ORZ sources Materials and methods Fish Sci (2011) 77:431–437 Table Pellets of rainbow trout formula (g) Ingredients Control diet ORZ diet Anchovy meal 600 600 Dextrin 104 104 50 50 Fish oil 100 100 Corn oil 100 100 Pregelatinized starch P-free mineral mix 10 10 Vitamin premixture 30 30 Choline chloride 5 Vitamin E (50%) 1 Gamma oryzanol Total 1,000 0.010 1,000 Table Extruded pellets of yellow tail diet (g) Ingredients Control diet ORZ diet Anchovy meal 540 540 Wheat flour 100 100 Pregelatinized starch 60 60 Lupin meal (dehulled) 60 60 180 20 180 20 Animals Fish oil Soybean meal Male C57BL/6j mice (7 weeks, an average weight of 22 g) were obtained from Clea Japan (Tokyo, Japan) They were maintained at 50% relative humidity and a 12-h light/dark cycle at 20–22°C Mice were given water and the diet with ORZ (120 mg/kg) ad libitum Two months later, soleus muscle and liver were collected from three mice for analysis All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Tokyo University of Marine Science and Technology About 50 individuals (approximately 300 g) of healthy rainbow trout Oncorhynchus mykiss were stocked in water tanks and fed two times per day (water temperature of rearing fish was about 15 ± 2°C) The fish were fed with the experimental diets supplemented with or without ORZ (10 mg/kg diet) (Table 1) After months, abdominal white muscle was collected and subjected to analysis Yellowtail Seriola quinqueradiata were reared in a local fish farm in Kagoshima prefecture, Japan, and were fed a control diet for over month and subsequently fed a diet containing ORZ diet (150 mg/kg diet, Table 2) about three times per week Five fish were caught every month and transported to the laboratory on ice within 48 h of being caught Abdominal white muscle and liver were collected and subjected to analysis Red sea bream Pagrus major were cultured in a local farm in Ehime prefecture, Japan, and were fed a control diet for over month and subsequently a diet containing Corn gluten meal 16 16 Mineral mix 10 10 Vitamin premixture 10 10 4 123 Rice bran Gamma oryzanol Total 1,000 0.150 1,000 Table Extruded pellets of red sea bream diet (g) Ingredients Anchovy meal Control diet ORZ diet 480 480 Shrimp meal 50 50 Wheat flour 110 110 Lupin meal (dehulled) 110 110 Fish oil 70 70 Soybean meal 45 45 Rice bran 70 70 Corn gluten meal 45 45 Mineral mix 10 10 Vitamin premixture 10 10 Gamma oryzanol Total 1,000 0.100 1,000 ORZ (100 mg/kg diet, Table 3) every day Five fish were caught every month and transported to the laboratory on ice within 48 h of being caught Abdominal white muscle and Fish Sci (2011) 77:431–437 433 liver were collected and subjected to analysis Wild fish that had been caught along the coast around the Oita prefecture were used for the control experiment (P \ 0.05) Body length and body weight of fish gradually increased during the experimental period Contents of ORZ in rainbow trout Measurement of ORZ content Muscle and liver were homogenized and extracted with chloroform/methanol solution for total lipid extraction according to the procedure of Bligh and Dyer [24] ORZ in total lipid was labeled with 0.5 mM 1-pyrenesulfonyl chloride (PSC, Molecular Probes) according to the method of Fujino et al [25] with slight modifications and separated through a reverse-phase HPLC system equipped with a Mightysil RP-18GP column (250 4.6 mm, lm, Kanto Chemical, Tokyo, Japan) as a solid phase Cholesteryl ferulate was used as an internal standard and the fluorescence of eluted pyrene-labelled compounds were detected at 330 nm of excitation and 390 nm of emission using a RF-10AXL (Shimadzu, Kyoto, Japan) Statistical analyses Rainbow trout body condition data were statistically analyzed between control and ORZ groups by paired Student’s t test Data for ORZ accumulation were evaluated using Steel-Dwass analyses The values with P \ 0.05 were judged significantly different Results Contents of ORZ in mice Table shows contents of ORZ in mice After months, ORZ was not detected in skeletal muscle of mice Contents of ORZ in liver were 0.17 lg/g liver No ORZ was observed in muscle and liver of control group Fish body condition Body lengths and body weights of the ORZ diet groups of rainbow trout, yellowtail, and red sea bream and of the rainbow trout control diet group are shown in Fig 1a–c In body weight, there were significant differences between the control and ORZ diet groups (306.9 ± 43.9 and 384.0 ± 57.1 g, respectively) of rainbow trout after months Table Contents of ORZ in mice (lg ORZ/g tissue) Tissue Control diet ORZ diet Skeletal muscle ND ND Liver ND 0.17 ND Not detected After months, contents of ORZ in the muscle of rainbow trout in the control and ORZ diet groups (37.3 ± 13.6, 98.0 ± 54.9 lg/g tissue) were significantly higher than initial groups (P \ 0.05) (Fig 2) We also evaluated the clearance of ORZ in vivo Administration of the control diet for month after the ORZ diet reduced ORZ levels almost to the control level Contents of ORZ in yellowtail Figure 3a shows the contents of ORZ in yellowtail muscle There were significant differences among initial levels (77.3 ± 22.9 lg/g tissue), month samples, and the later samples (P \ 0.05) As shown in Fig 3b, the ORZ levels in liver after and months (499.7 ± 145.3 lg/g tissue, P \ 0.01; 283.4 ± 82.4 lg/g tissue, P \ 0.05, respectively) were significantly higher than initial levels (105.8 ± 14.8 lg/g tissue) Contents of ORZ in red sea bream As shown in Fig 4a, the ORZ levels in red sea bream muscle throughout the experimental period were significantly higher than initial levels (139.0 ± 21.6 lg/g tissue, P \ 0.05) The ORZ levels of fish fed the ORZ diet were significantly higher than the ORZ levels of wild red sea bream (55.6 ± 17.2 lg/g tissue, P \ 0.05, data not shown) Figure 4b shows the contents of ORZ in red sea bream liver There were significant differences among initial levels (255.8 ± 76.8 lg/g tissue) and 1- and 2-month samples (656.9 ± 82.7 lg/g tissue, P \ 0.01; 611.9 ± 365.1 lg/g tissue, P \ 0.05) The levels in the 1-month sample were significantly higher than the levels in the wild fish (300 ± 21.5 lg/g tissue, P \ 0.05, data not shown) Discussion We recently reported that ORZ enhances adiponectin secretion by the inhibition of NF-jB activation and ameliorates type diabetes in mice [20, 26] In the present study, an investigation of the in vivo energy metabolism was carried out and revealed that body weight gain was observed in rainbow trout, probably because of conserved protein used for energy production (Fig 1) Although mouse muscle and liver hardly accumulated ORZ, the muscle and liver of fish including rainbow trout, yellow tail, and red sea bream accumulated high amounts of ORZ 123 434 35 500 a Control ORZ 30 Control * ORZ 400 Body length Body weight 25 300 20 15 200 Body weight (g) Body length (cm) Fig Body length and body weight of rainbow trout (a), yellowtail (b), and red sea bream (c) (n = 5) Closed circles indicate body length of the control group, open circles body length of the ORZ group in a The asterisk denotes that there are significant differences between control and ORZ groups (*P \ 0.05) by multiple comparisons Fish Sci (2011) 77:431–437 10 100 0 Initial month month 100 8000 b Body length Body weight 80 60 4000 40 Body weight (g) Body length (cm) 6000 2000 20 0 Initial month month month month month 1400 40 Body length c Body weight 30 1000 800 20 600 Body weight (g) Body length (cm) 1200 400 10 200 0 Initial These findings suggest that teleost positively accumulates ORZ in several organs The accumulation of ORZ in the muscle and liver of yellowtail was confirmed after feeding ORZ for months However, a notable change was not observed after feeding ORZ for months, which indicates that 200–300 lg/g tissue might represent a level of saturation in the accumulation of ORZ for the yellowtail Significant ORZ accumulation was also observed in the red 123 month month month sea bream muscle after ORZ feeding for months, while ORZ feeding for month induced ORZ accumulation in the liver These results suggest that ORZ is accumulating in the muscle via the liver and circulation, although ORZ levels in circulation were not determined in the present study because they were very low Relatively low levels of ORZ were detected in wild red sea bream, although red sea bream is carnivorous Wild red sea bream might 180 160 140 120 b** 100 80 60 a* 40 a** a** 20 Control ORZ Control ORZ Control ORZ Initial month month Oryzanol content in yellowtali muscle µg oryzanol / g tissue Fig Accumulation of ORZ in rainbow trout muscle The mean values are presented with standard deviation (n = 5) The asterisks denote that there are significant differences (**P \ 0.01, *P \ 0.05) from the initial control and ORZ value, respectively The different letters represent significant differences (P \ 0.05) by multiple comparisons 600 a * 500 * * * 400 1200 a * * 400 * 300 200 100 Initial b month ** month month month month * 1000 800 600 400 200 month Fig Accumulation of ORZ in red sea bream muscle (a) and liver (b) The mean values are presented with standard deviation (n = 5) and were significantly different from the initial values with **P \ 0.01, *P \ 0.05 by multiple comparisons 200 100 Initial Oryzanol content in yellowtali liver µg oryzanol / g tissue 500 Initial 300 700 Oryzanol content in red sea bream muscle µg oryzanol/ g tissue 435 Oryzanol content in red sea bream liver µg oryzanol/ g tissue Oryzanol content in rainbow trout muscle µg oryzanol / g tissue Fish Sci (2011) 77:431–437 b month month month month ** month * 600 500 400 300 200 100 Initial month month month month month Fig Accumulation of ORZ in yellowtail muscle (a) and liver (b) The mean values are presented with standard deviation (n = 5) and were significantly different from the initial values with **P \ 0.01, *P \ 0.05 by multiple comparisons accumulate a small amount of ORZ via the food chain because seaweed, such as laminaria, also contain ORZ [27] As shown in Fig 2, feeding of the control diet following the ORZ diet tended to reduce ORZ levels, suggesting that ORZ is gradually excreted from or metabolized in rainbow trout muscle for about month The absorption and excretion pathways for ORZ in teleosts are, however, still unknown In mammals, the intestine has a barrier that prevents the absorption of plant sterols and their derivatives, although the structures of plant sterols are very similar to that of cholesterol [28] This was confirmed in the present which showed that ORZ was hardly accumulated in mouse tissues (Table 4) Plant sterols differ from cholesterol only by an additional methyl group (campesterol), ethyl group (sitosterol) at the C24 position, an additional double bond at the C22 position (stigmasterol), or by an additional methyl group and double bond at the C22 position (brassicasterol) Both cholesterol and plant sterols are internalized by intestinal mucosa cells via the Niemann-Pick C1-Like1 (NPC1L1) transporter [29] Cholesterol is transported to the endoplasmic reticulum, where it is esterified by the action of acyl-CoA:cholesterol O-acyltransferase (ACAT2) for incorporation into chylomicrons [30] However, plant sterols are poor for ACAT2 123 436 substrates and hence are transported back to the luminal membrane to be re-secreted into the lumen of the intestine by the heterodimeric ATP-binding cassette (ABC) transporters g5 and g8 [31] ABCg5 and ABCg8 are present at the apical membrane of enterocytes and are also expressed in liver [32] In the liver, the ABCg5/8 transporter mediates efflux of plant sterols into bile [33, 34] Recent studies have demonstrated that increased circulating levels of plant sterols, as a result of intake of a plant sterol-enriched diet in wild-type mice or as a consequence of ABCg5 or ABCg8 deficiency, are associated with elevated levels of plant sterols in the brain [35] The classic studies demonstrating that plant sterols were observed in fish muscle [36, 37] make us believe that these barrier systems for plant sterols would be immature in fish Although transporters conveying ORZ are unknown even in mammals, it is thought that ORZ could be possibly transported via similar transporters 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biliary cholesterol excretion J Biol Chem 278:48275–48282 34 Plo¨sch T, Blocks VW, Terasawa Y, Berdy S, Siegler K, Sluijs FVD, Kema IP, Groen AK, Shan B, Kuipers F, Schwarz M, Schwartz M (2004) Sitosterolemia in ABC-transporter G5-deficient mice is aggravated on activation of the liver-X receptor Gastroenterology 126:290–300 35 Jansen PJ, Lu¨tjohann D, Abildayava K, Vanmierlo T, Plo¨sch T, Plat J, Bergmann K, Groen AK, Ramaekers FCS, Kuipers F, Mulder M (2006) Dietary plant sterols accumulate in the brain Biochim Biophys Acta 1761:445–453 36 Kritchevsky D, Tepper SA, DiTullo NW, Holmes WL (1967) The sterols of seafood J Food Sci 32:64–66 37 Morris RJ, Ballantine JA, Roberts JC, Lavis A (1982) The sterols of some marine teleosts Comp Biochem Physiol 73B:481–484 123 Fish Sci (2011) 77:439–446 DOI 10.1007/s12562-011-0344-9 ORIGINAL ARTICLE Food Science and Technology Effect of setting conditions on mechanical properties of acid-induced Kamaboko gel from squid Todarodes pacificus mantle muscle meat Bodin Techaratanakrai • Moemi Nishida Yuki Igarashi • Manabu Watanabe • Emiko Okazaki • Kazufumi Osako • Received: 11 December 2010 / Accepted: March 2011 / Published online: April 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Squid Todarodes pacificus suwari gels, set at various temperatures and times, and acid-induced kamaboko gel, which was prepared by soaking suwari gel in 5% acetic acid for 20 h, were studied to evaluate the mechanical properties that are affected by setting conditions Unset squid meat paste did not form a gel when soaked in acetic acid The breaking strength of both suwari gel and acid-induced kamaboko gel showed a tendency to increase with setting temperature and time SDS-PAGE analysis of suwari gel and acid-induced kamaboko gel, which were set at various temperatures and times, showed that myosin heavy chain (MHC) was observed at 30°C only for the first hour The intensity of the MHC band at 30°C gradually decreased with setting time, while the intensity of the polymer band gradually increased with setting time These results suggest that the protein-protein bonds in suwari gel affect the final texture of acid-induced kamaboko gel Based on the analysis of the mechanical properties, and in consideration of the fact that the purpose of this experiment was to reduce energy usage, the best setting condition was determined to be 40°C for h Keywords Suwari gel Á Acid-induced kamaboko gel Á Squid Á Setting conditions Á Mechanical properties B Techaratanakrai Á M Nishida Á Y Igarashi Á M Watanabe Á E Okazaki Á K Osako (&) Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan e-mail: osako@kaiyodai.ac.jp Introduction Japanese common squid Todarodes pacificus is one of the most important fishery products in Japan Approximately 500,000 tons of squid is caught in Japanese waters every year, and the catch is estimated as 4.5% of that generated by the entire Japanese fishery [1] Approximately 40% of the squid supply is marketed as fresh or frozen for sashimi, sushi, and home-cooking The remainder is processed into a wide variety of products, including ika-kamaboko (squidbased kamaboko), in response to consumer demand [2] In general, kamaboko is produced via a multi-step process First, surimi is mixed with salt to induce dissociation of actomyosin [3] Next, to improve kamaboko gel strength, which is one of the characteristics associated with high-quality surimi products [4], functional additives such as egg white are added [5] Additionally, the setting process is often optimized by incubation of the surimi at temperatures lower than 40°C in order to enhance gel strength [6] The resultant gel product is called suwari gel [7] Finally, the suwari gel is boiled, steamed, or baked at more than 80°C to produce kamaboko In industrial manufacturing, the heating process involves high costs, both in terms of fixed and variable costs, such as those from electrical and other utilities At the same time, a method that does not require a heating process is available for making kamaboko This method has been traditionally used for making fish meat gel in the Kyushu region of Japan, generating a product called sujime-kamaboko [8] The principle behind making this product is similar to typical kamaboko; however, in the case of sujime-kamaboko, suwari gel is soaked in vinegar or an acetic acid solution at a low temperature as the final cooking step instead of heating [9] Thus, sujime-kamaboko can be called acid-induced kamaboko gel 123 440 The setting step is one of the most important steps in producing a surimi-based product for the preparation of suwari gel During setting, it is generally thought that the strength of the gel matrix is mostly derived from intermolecular interactions, although covalent bonds resulting from transglutaminase action have been reported [10] From investigations into the effect of the initial network formed during setting on the texture of set and cooked gels, it was concluded that there is a range of optimum setting temperatures and times that ensures kamaboko gels have good textural characteristics [10], and these optimum conditions vary according to the fish used [11] The gel-forming ability of cephalopod muscle is very low due to its high proteolytic ability and low endogenous transglutaminase activity [12] It has been reported that metalloprotease is the major protease of Japanese common squid mantle muscle [13] and is characterized by optimum pH activity at 7.0 and optimum temperature of 40°C [14] Usually, sodium chloride is used in kamaboko production for impasting the fish muscle However, in the case of squid mantle muscle meat, sodium chloride promotes the protease activity and has been reported to cause very poor texture in kamaboko [15] Therefore, the use of organic salts as solubilizing agents, such as sodium gluconate (a known chelator), is preferred to sodium chloride [15] Shortage of endogenous transglutaminase results in a gel of poor texture due to an insufficient amount of enzyme to catalyze the formation of a covalent crosslinking network within the gel structure; therefore, the gel does not set during the setting period resulting in texture problems in kamaboko [12] The use of microbial transglutaminase is another possible means of improving gelation Moreover, some studies have reported that protease inhibitors and microbial transglutaminase can be advantageously combined in fish species with poor gelforming ability [16] In the present study, the first aim was to demonstrate the necessity of using microbial transglutaminase and replacing sodium chloride with sodium gluconate in acid-induced gelation of squid mantle muscle meat Secondly, the effect of setting conditions on the quality of acid-induced kamaboko gel was investigated in terms of certain mechanical properties and SDS-PAGE patterns of suwari gel and acidinduced kamaboko gel from squid meat Materials and methods Samples Japanese common squid Todarodes pacificus caught from the Japanese Sea off the coast of Tsushima, Nagasaki, in 123 Fish Sci (2011) 77:439–446 July 2009, was purchased from the local market within 12 h of capture, packed in ice, and brought to the laboratory under ice storage conditions on the day following the catch The average weight of the squid was approximately 200 g The squid was degutted and skinned at temperatures below 10°C in order to get the mantle muscle meat The proximate components of raw squid mantle muscle meat were moisture 78.2 ± 0.05%, crude protein 16.5 ± 0.01%, total lipids 1.01 ± 0.14%, crude ash 1.59 ± 0.03%, and crude carbohydrate 2.65% The mantle muscle meat was then minced with a meat chopper (Meat Chopper M-22A, Nantsune, Osaka, Japan) Sucrose was added to the minced mantle muscle meat and mixed well at a final concentration of 5% (w/w) The product of this step is called surimi About 100 g of surimi was then packed into a polystyrene bag and kept at -50°C until use The effects of sodium gluconate and microbial transglutaminase on suwari gel and acid-induced kamaboko gel formation The method of acid-induced kamaboko gel preparation was modified from Abe et al [8] There were four conditions to be studied For the first condition, frozen surimi was thawed, chopped, and then tempered for 10 using a grinding machine (Ishikawa-shiki no 20, Ishikawa Kojo, Tokyo, Japan) A 1% sodium chloride solution was added and homogenized by the grinding machine for 15 After that, 1% microbial transglutaminase (ActivaÒ TG-K, Ajinomoto, Tokyo, Japan) and 3% powdered egg white (Nippon Colloid, Tokyo, Japan) were added, and these ingredients were homogenized for 15 more During these steps, the temperature of the squid meat was controlled below 10°C The squid paste was shaped in a stainless steel vessel with a 32 mm inside diameter and 30 mm length The squid meat paste was put in a water bath (Eyela NTT-2400, Tokyo Rikakikai, Tokyo, Japan) at 40°C for h to set the meat paste The product of this step is called suwari gel The suwari gel at each setting condition was removed from the vessel, soaked in 5% acetic acid, and put in the water bath (Fine Thermo F-002DA, TGK, Tokyo, Japan) at 15°C for 20 h The final product is called acid-induced kamaboko gel For conditions 2–4, the preparation was the same but differed in the type and amount of salt and amount of microbial transglutaminase In condition 2, sodium chloride was not added In condition 3, sodium chloride was replaced with sodium gluconate Condition was similar to condition except that microbial transglutaminase was not added The appearance of suwari gel and acid-induced kamaboko gel obtained under these conditions was studied Fish Sci (2011) 77:439–446 The effects of setting condition of suwari gel and acid-induced kamaboko gel The preparation of suwari gel and acid-induced kamaboko gel was the same as described above Addition of sodium chloride, sodium gluconate, and/or microbial transglutaminase was determined based on the results of the previous step Setting temperatures were 30, 40, and 50°C and setting times were 0, 0.5, 1, 3, 6, and 12 h pH of suwari gel and acid-induced kamaboko gel was measured by homogenizing 10 g suwari gel or acidinduced kamaboko gel with 90 g water with a homogenizer (Heidolph Diax 600, Heidolph Instruments, Schwabach, Germany) and measuring with a microprocessor pH meter (pH211, Hanna InstrumentÒ, Woonsocket, RI, USA) Gels were measured for their breaking strength (g) and breaking deformation (cm) with a rheometer (Rheoner RE3305-I, Yamaden, Tokyo, Japan) equipped with spherical plunger (diameter mm) with a mm/s raising rate of the sample table Every measurement was repeated five times, and the mean values were presented Samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared using a method similar to suwari gel and acid-induced kamaboko gel preparation step Instead of shaping in the stainless steel vessel, the squid paste was shaped into a thin film pattern (8.5 2.0 0.2 cm) to guarantee that the acid penetrated the gel thoroughly The samples were solubilized with SDS-urea solution containing 2% SDS, M urea, 50 mM Tris-HCl (pH 8.81), and 2% mercaptoethanol by heating at 100°C for and dialyzed overnight in SDS-urea solution that did not contain mercaptoethanol SDS-PAGE was conducted as described by Laemmli [17] using 7.5% separating polyacrylamide gels (PAGELÒ, Atto, Tokyo, Japan) Gels were stained with Coomassie brilliant blue R250 Images of the scanned gel were analyzed by the ImageJ software (available at http://rsbweb nih.gov/ij/) The areas under the myosin heavy chain peak of suwari gel and acid-induced kamaboko at each setting 441 condition were compared with unset suwari gel and acidinduced kamaboko gel and calculated as the percent of relative content of myosin heavy chain Results The effects of sodium gluconate and microbial transglutaminase on suwari gel and acid-induced kamaboko gel formation The appearance of suwari gels and acid-induced kamaboko gels is presented in Table Suwari gels and acid-induced kamaboko gels with the addition of either sodium chloride (condition 1), microbial transglutaminase (condition 2), or sodium gluconate (condition 4) did not set In contrast, suwari gel with the addition of sodium gluconate and microbial transglutaminase (condition 3) was observed to form a very weak gel In addition, acid-induced kamaboko gel under condition formed a soft gel The effects of setting condition on suwari gel and acid-induced kamaboko gel The pH of suwari gel and acid-induced kamaboko gel was 6.54 ± 0.13 and 3.97 ± 0.28, respectively After soaking in acetic acid for 72 h, squid meat paste (setting time = 0) was not set and was of very poor quality in terms of both visual appearance and texture The mechanical properties of squid meat paste both before and after soaking in acid could not be measured; therefore, the results of the unset suwari gel and acid-induced kamaboko gel are not shown in the figures below As shown in Fig 1, the breaking strength of suwari gel increased with increasing temperature and setting time (30°C, 8.64–31.04 g; 40°C 55.68–84.61 g; 50°C 129.70–188.54 g) Assessment of the breaking deformation of suwari gel, as illustrated in Fig 2, gave different results from breaking strength At 30 min, the suwari gel at Table Appearance of suwari gel and acid-induced kamaboko gel prepared under various conditions Condition 1% sodium chloride ? 1% microbial transglutaminase Gel type Appearance Suwari gel Gel was not set Acid-induced kamaboko gel Gel was not set Gel was not set 1% microbial transglutaminase Suwari gel Acid-induced kamaboko gel Gel was not set 1% sodium gluconate ? 1% microbial transglutaminase Suwari gel Gel was very soft 1% sodium gluconate Acid-induced kamaboko gel Gel was soft Suwari gel Gel was not set Acid-induced kamaboko gel Gel was not set 123 442 Fish Sci (2011) 77:439–446 Fig Effect of setting time on breaking strength of suwari gel Data are shown as mean ± standard deviation The gel at h is not shown because it could not be measured 40°C showed the highest breaking deformation, while the others showed little or no difference in breaking deformation with increasing time The acid-induced kamaboko gel set at 30°C had a slimy surface, whereas this was not observed in the acid-induced kamaboko gels set at 40 and 50°C The breaking strength of the acid-induced kamaboko gels set at the various temperatures increased with increasing setting time, with the exception of the gel set at 50°C for 12 h, which was slightly less strong than that set at 50°C for h, as shown in Fig (30°C, 64.96–83.28 g; 40°C, 100.48–132.48 g; 50°C, 155.65–188.54 g) The breaking deformation of acid-induced kamaboko gel is shown in Fig After soaking in the acetic acid solution, the breaking deformation of the gel set at 30°C was greatest regardless of setting time, whereas the values did not differ among the gels set at 40 and 50°C, regardless of heating time SDS-PAGE analysis of suwari gel and acid-induced kamaboko gel is illustrated in Fig In both suwari gel and acid-induced kamaboko gel, at a setting temperature of 30°C and a setting time of less than h, MHC bands decreased in intensity with setting time However, at a setting temperature of 30°C and a setting time greater than h, and at the setting temperatures of 40 and 50°C, MHC bands were not visually detected; however, polymer bands were clearly observed Moreover, there was a band between myosin heavy chain and paramyosin at 30°C This band and the paramyosin band also decreased in intensity with a longer setting time, similar to that seen with MHC Figure illustrates the changes in myosin heavy chain content It was also shown that myosin heavy chain content was reduced with setting time The myosin heavy chains of suwari gel set at 50°C for longer than h and acid-induced kamaboko gel set at 40°C for longer than h and at 50°C for longer than h were not detected 123 Fig Effect of setting time on breaking deformation of suwari gel Data are shown as mean ± standard deviation a Setting at 30°C; b setting at 40°C; c setting at 50°C The gel at h is not shown because it could not be measured Discussion Sodium gluconate and microbial transglutaminase were both necessary for preparing acid-induced kamaboko gel While sodium chloride promotes protease activity in squid mantle muscle meat, sodium gluconate inhibits this activity via a chelating mechanism [15] Microbial transglutaminase also improves gelation by catalyzing the formation of extensive covalent crosslinking, chiefly involving myosin heavy chain [12] Sodium gluconate, as a protease inhibitor, and microbial transglutaminase must be combined in Fish Sci (2011) 77:439–446 443 Fig Effect of setting time on breaking strength of acid-induced kamaboko gel Data are shown as mean ± standard deviation The gel at h is not shown because it could not be measured order to produce suwari gel Moreover, it was clearly observed that if the suwari gel did not set, the acid-induced kamaboko gel would not set either This suggests that the setting step is essential for the production of acid-induced kamaboko gel from squid mantle muscle meat During the setting step, myofibrillar proteins establish links with adjacent molecules by covalent bonding, compacting the gel structure [18] via transglutaminase catalysis, and leading to the strengthening of the suwari gel When soaking suwari gel in acetic acid, proteins were denatured or unfolded, which occurred subsequent to the formation of the network structure [19] At 30°C, suwari gel was not completely set in the studied time; therefore, the breaking strength of suwari gel at 30°C was very low After soaking in acetic acid solution, the acid-induced kamaboko gel showed approximately 50 g higher breaking strength than suwari gel at each setting time; however, the appearance was poor with a slimy surface This may have been due to the myofibrillar protein in suwari gel heated at 30°C not being set completely, thereby unfolding due to the acid effect and subsequently refolding into a nonnative structure due to the effect of the acid anion (acetate ion) [20] Acid-induced kamaboko gel from suwari gel set at 40°C did not have a slimy surface and also showed approximately 50 g higher breaking strength than suwari gel at each setting time On the other hand, the changes in breaking strength at 50°C were obviously smaller than the rest It was possible that the muscle protein was denatured and aggregated during setting at 50°C; therefore, the formation of the bonds induced by acetic acid may be hard to achieve Moreover, excessively high temperature and a long setting time caused a reduction in gel breaking strength of acid-induced kamaboko gel This decrease may be due to destruction of the gel matrix network, associated with the modori phenomenon [10] caused by cysteine protease [21] Taking into consideration the breaking Fig Effect of setting time on breaking deformation of acidinduced kamaboko gel Data are shown as mean ± standard deviation a Setting at 30°C; b setting at 40°C; c setting at 50°C The gel at h is not shown because it could not be measured strength result only, the best setting condition would be 50°C for h Increasing the setting temperature and time caused a decrease in the breaking deformation of suwari gel This may be because the gel matrix tightened and formed a closer network [22], leading to a reduction in breaking deformation However, at 30°C the gel was not completely set; therefore, the breaking deformation of suwari gel at that temperature was low, similar to its breaking strength During the soaking of suwari gel in acetic acid solution, the acid penetrated the gel structure, forming a more compact protein network and thereby conferring more brittle 123 444 Fish Sci (2011) 77:439–446 Fig SDS-PAGE patterns of myofibrillar protein in suwari gel and acid-induced kamaboko gel at various setting times and setting temperatures a Suwari gel; b acid-induced kamaboko gel characteristics [10] Taking into consideration the breaking deformation result only, the best setting condition would be at 30°C for h Taking into consideration both the breaking strength and breaking deformation results, the best setting condition for acid-induced kamaboko gel from squid mantle muscle meat was 40°C for h Acid-induced kamaboko gel at 30°C gave high breaking deformation; however, as previously mentioned, the appearance was very poor and could not be used in production Although the acid-induced kamaboko gel at 50°C gave a higher breaking strength than acid-induced kamaboko gel at 40°C, and although their breaking deformation values were similar, the high temperature was in conflict with the purpose of the study, which was to reduce energy costs Therefore, the gel at 50°C was not selected A setting time of h was chosen 123 because the resultant acid-induced kamaboko gel had the highest breaking deformation, while the breaking strength did not differ from gels set for longer times The SDS pattern suggested that crosslinking of the myosin heavy chain occurred continuously during setting It was observed that a decrease in MHC band intensity was inversely related to the intensity of the polymer band A high degree of protein aggregation is mediated by transglutaminase activity [12] Microbial transglutaminase activated the crosslinking of MHC [23], which is one of the factors involved in the gelation of kamaboko [24] Because the optimum temperature of microbial transglutaminase is 50°C [25], protein aggregation occurring in suwari gel set at 30°C was slower than that of gels set at 40 and 50°C There are several reports that revealed that the absence of MHC bands and the presence of polymer bands were Fish Sci (2011) 77:439–446 Fig Changes in the content of myosin heavy chain in suwari gel and acid-induced kamaboko gel at various setting times and setting temperatures a Suwari gel; b acid-induced kamaboko gel related to the mechanical properties of the gel [23, 26, 27] During setting, crosslinking of myosin was established and led to MHC band reduction and polymer band increase, as revealed by SDS-PAGE analysis Comparing changes in breaking strength, which increases with setting time and temperature, to myosin heavy chain content, which decreases with setting time and temperature, suggests that lower myosin heavy chain content is associated with higher breaking strength in suwari gel and acid-induced kamaboko gel Acknowledgments This study was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology References Ministry of Agriculture, Forestry and Fisheries (2008) Suisanhakusho (in Japanese) Ministry of Agriculture, Forestry and Fisheries, Tokyo Court WG (1980) Japan’s squid fishing industry Mar Fish Rev 42:1–9 445 Mackie IM (1993) The effect 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Education, Tokyo Gakugei University, 4-1-1 Nukuikitamachi, Koganei, Tokyo 184- 850 1, Japan Y Kawaguchi Division of Ecosystem Design, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-josanjima, Tokushima 880- 850 6, Japan M Ichimura Shibetsu Salmon Museum, 1-1 Kita 1-Jo Nishi 6-Chome, Shibetsu, Shibetsu-gun, Hokkaido 086-163 1, Japan K Edo Monuments and Sites Division, Agency... Hamduk, Jocheon, Jeju Special Self-Governing Province 6 95- 81 4, Republic of Korea genes in certain tissues is partially regulated in a circadian manner Keywords Cloning Á Day–night variations Á Food availability Á Rabbitfish Á Quantitative real-time PCR Á Temperature Introduction There are two types of thyroid hormones (THs ), namely, 3 ,5 ,3 0 ,5 0 -tetraiodothyronine (T4) and 3 ,5 ,3 0 -triiodothyronine (T3 ),. .. 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