best correspondence between the actual EPT and coagulation temperature of the filtrates was found ... bovine serum albumin (Sigma Chemical Co., St Louis,.
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2000; 66: 153–160
Original Article
Coagulation test for determining end-point temperature of heated blue marlin meat MUSLEH UDDIN, SHOICHIRO ISHIZAKI AND MUNEHIKO TANAKA* Laboratory of Food Processing, Department of Food Science and Technology, Tokyo University of Fisheries, Konan, Minato, Tokyo 108-8477, Japan SUMMARY: Applicability of a coagulation test was evaluated for the determination of the previous heat condition of heated blue marlin meat. Meat blocks were heated at different temperatures between 50 and 70°C. Proteins were extracted with different concentrations of NaCl, then subsequently subjected to the coagulation test. The coagulation method was able to determine the end-point temperature (EPT) of heated blue marlin meat within a range of 1–2°C, up to 67°C. The best correspondence between the actual EPT and coagulation temperature of the filtrates was found when proteins were extracted with 0.9% saline solution. Within the range of 50–67°C, sample preparation methods and holding times had no significant effect on the coagulation temperature of the filtrates. Protein bands of the filtrates on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel gradually disappeared with increasing temperature. One band with a molecular weight of about 31 kDa was detected in 67°C-, but not in 70°C-heated meat. This protein component was responsible for coagulation at 67°C and was found to be lactate dehydrogenase from the analyses by gel filtration, SDS-PAGE, and enzyme activity. KEY WORDS: blue marlin, coagulation, end-point temperature, lactate dehydrogenase, sodium dodecylsulfate-polyacrylamide gel electrophoresis.
INTRODUCTION Food-borne pathogens are a potential health threat to all consumers and measures to prevent microbial growth are of primary concern to retailers, food processors, consumer advocacy groups, and regulatory agencies. Consequently, most food products receive some type of heat treatment by commercial processes prior to marketing and/or by domestic cooking prior to consumption. Inadequate cooking is commonly cited as a contributing factor in food-borne disease outbreaks caused by meat and poultry products.1 Many outbreaks result from undercooking at food service and retail outlets where the time and temperature of processing do not need to be adequately documented. Heat treatment of food products is important not only for reducing the risk of infection from heat-labile pathogens,2 but also for producing a palatable product and improving shelf-life. The
*Corresponding author: Tel: 54630611. Fax: 54630627. Received 25 June 1999.
method to determine the previous heat conditions of processed products would be useful and beneficial to the food processing industry. Townsend and Blankenship reported an overview of methods used for determining the maximum temperature of previously heat-treated meat.3 Numerous attempts have been made to develop tests capable of determining previous heat treatment of processed products, but most of the methods used to verify end-point temperature (EPT) are time consuming, subjective, variable and lacking in accuracy for predicting the actual heating end-point achieved during processing.4, 5 The reliability of these methods is critical with regard to food safety, corporate liability, and regulatory substantiation of prior heat treatment, and thus their shortcomings pose serious concerns for the safety of precooked processed products. Therefore, the United States Department of Agriculture (USDA) and other groups proposed a simple, rapid, and easy test with minimum subjectivity which can accurately determine EPT of processed foods. For meat products, tests on residual enzyme activity or on extractability and coagulation properties of proteinaceous materials6,7 gave indication of heat treatment
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within a small temperature range. Bovine catalase test was developed by Eye8 for the detection of the underprocessing of rare roast beef and cooked beef. Keeton and Morris studied the effects of curing on triosephosphate isomerase (TPI), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activity in turkey meat.9 They found that TPI lost its activity in fresh meat between 70 and 72°C. Activities of MDH and LDH in fresh turkey meat were both observed to decline sharply between 72 and 74°C, while in cured turkey breast the activity was lost over a broader range of 70 to 74°C. Lee et al. employed sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the study of qualitative changes in electrophoretic patterns of low-salt soluble proteins10 to assess the final cooking temperature.11 Differential scanning calorimetry (DSC) has been also used to investigate the denaturation of meat proteins11–13 and to estimate the maximum cooking temperature of meat products.14,15 Wang et al. recently suggested that proteins such as bovine serum albumin, glyceraldehyde-3phosphate dehydrogenase (GAPDH), phosphoglycerate mutase, and TPI might be useful to verify the processing temperature of ground beef patties.16 Although a large number of studies have been conducted to determine EPT of processed beef, chicken, pork and other meat products, similar studies on processed marine products are almost negligible. Since marine products have an ever-growing role as a human food source, it becomes even more important to achieve adequate heat treatment which prevents hazardous food-borne diseases2 and protects loss of food quality. The amount of undenatured proteins in a heated product and their characteristics have been used as the basis of a method for determining the EPT achieved. The coagulation test (USDA, Chemistry Laboratory Guidebook)7 is based on the principle that when proteins are heated, denaturation and coagulation occur progressively until all the proteins become insoluble and are precipitated out from the solution. The objective of this study was to evaluate the applicability of a coagulation method for determining EPT of blue marlin meat heated at different temperatures with different holding times. In addition, other methods such as SDS-PAGE and enzyme activity determination were also employed to reveal the relationship between EPT and coagulation temperature.
MATERIALS AND METHODS Fish meat Blue marlin Makaira mazara which had quickly been frozen to –40°C on board immediately after catch and stored at –40°C was obtained from Misaki, Kanagawa Prefecture, Japan. The proximate composition and protein composition of blue marlin meat have been reported previously by Wahyuni et al.17
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Heat treatment Frozen blocks of blue marlin meat were thawed overnight at 5°C. Ground meat was prepared with a Kitchen Aid FMI-K5SS grinder (Kitchen Aid Inc., MI, USA) through a grinder plate with 4.7 mm holes. Homogenized blue marlin meat was prepared with a National MK-K75 food processor (National Electric Co., Osaka, Japan). Eighty grams each of meat block (40 ¥ 40 ¥ 40 mm), ground meat, or homogenized meat was wrapped in polyvinylidene chloride film. Samples were immersed in a stirred water bath preheated to one of the selected temperatures, and removed when the temperature of the samples indicated by thermocouples reached that of the water bath. Thermocouples (copper-constantan), connected to a recorder (Thermodac EF, Model 5020A; Eto Denki Co., Tokyo, Japan), were positioned at the geometric centers of the samples. Heating temperatures were selected between 50 and 75°C. It took about 20 min for the geometric centers to reach the selected heating temperatures. Samples were also heated for different holding times: 0, 5, 15, and 30 min. When the center of the meat mass attained the temperature of the water bath, the sample was considered to be at time zero and removed from the water bath. Three additional samples were maintained at the selected temperatures for either 5, 15, or 30 min. After reaching the specified temperature and holding time, the samples were immediately cooled in ice-cold water and stored at –40°C until tested.
Preparation of filtrate After the heat treatment, proteins were extracted from 50 g of each sample with 100 mL of distilled water, 0.4%, 0.9%, or 1.5% NaCl solution by homogenizing at 12 000 rpm for 1 min using a homogenizer (Model HG30; Hitachi Co., Tokyo, Japan). The homogenates were allowed to stand for 20 min at room temperature. This primary extract was filtered in accordance with the standard ‘Coagulation Test’ procedure7 proposed by Food Safety and Inspection Service (FSIS) through No. 1 filter paper (Toyo Roshi Co., Tokyo, Japan) in a Buchner funnel with the aid of vacuum. The filtrate was refiltered through No. 5 filter paper (Toyo Roshi Co.), and then through a glass filter (3G3, Hario Co.) layered with about 8 mm of Celite No. 545 (Kokusan Chemical Works Co., Tokyo, Japan). This procedure gave a visually clear filtrate, which was used for determining coagulation temperature. pH values of the primary extracts and of the filtrates were determined with a pH meter (Model HM-30V; Toa Electronics Ltd, Tokyo, Japan). Protein concentration of the filtrates was determined by the biuret procedure of Gornall et al.18 Crystalline bovine serum albumin (Sigma Chemical Co., St Louis,
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MO, USA) was used for the preparation of the standard curve.
Determination of coagulation temperature Coagulation temperature of the filtrates was measured by the ‘Coagulation Test’ procedure.7 A test tube containing an aliquot of 10 mL filtrate was placed and clamped into the position in the water bath. Thermocouples (copper-constantan) connected to a recorder (Thermodac EF, Model 5020A; Eto Denki Co.) were placed in the center of the test tubes. Then the water bath was heated at a rate to give a difference of 1.0–1.5∞C between the water bath and the filtrate temperature. The temperature at which the first sign of cloudiness appeared was noted and considered to be EPT to which the product had been processed, since the coagulation temperatures were well coincided with EPT. The determination for each sample was replicated three times.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis (SDS-PAGE) Sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed by using the method of Laemmli.19 The amount of protein applied was 20 µg per lane. Electrophoresis was carried out on a Biocraft vertical gel electrophoresis apparatus using 10% polyacrylamide gels at a constant current of 10 mA with 0.05 M Tris0.384 M glycine-0.1% SDS (pH 8.3) as a running buffer. A molecular weight standard mixture (Sigma Chemical Co.) composed of bovine serum albumin (66 kDa), egg albumin (45 kDa), GAPDH (36 kDa), bovine carbonic anhydrase (29 kDa), bovine pancreas trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa), and bovine milk a-lactalbumin (14.2 kDa) was used. Proteins were stained with Coomassie brilliant blue R250 (Wako Pure Chemical Industries Ltd, Tokyo, Japan), and scanned at 580 nm with a dual wavelength flying spot scanning densitometer (Type CS-9300 PC; Shimadzu Corp., Kyoto, Japan). Native polyacrylamide gel electrophoresis (7.5–15% gradient) was also performed on a Biocraft vertical gel electrophoresis apparatus at a constant current of 15 mA for 12 h with 0.05 M Tris-0.384 M glycine (pH 8.3) as a running buffer. Proteins were stained with Coomassie brilliant blue R250.
Gel filtration chromatography An aliquot of 5 mL filtrate was subjected to Sephadex G100 gel filtration column chromatography (2.8 ¥ 65 cm).
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Elution was performed using 0.066 M carbonate buffer (pH 10) containing 0.2 M NaCl at a flow rate of 0.4 mL/min. Absorbance was monitored at 280 nm. The standard proteins used for calibration were human gglobulin (160 kDa), bovine serum albumin (67 kDa), and ovalbumin (45 kDa). The void volume was determined with blue dextran 2000 (Sigma Chemical Co.).
Localization and determination of lactate dehydrogenase activity After native PAGE was achieved, LDH band was visualized by incubating the gel in LDH activity staining solution as described by Markert and Faulhaber.20 Lactate dehydrogenase activity was assayed spectrophotometrically21 with continuous monitoring of the decrease in absorbance at 340 nm due to oxidation of NADH using a UV-visible recording spectrophotometer (UV-160; Shimadzu Corp.). Activity of LDH in international units was calculated according to Stalder et al.21 GADPH activity was determined by the method of Krebs.22 In order to determine the thermal stability of LDH, the LDH fractions obtained by Sephadex G-100 gel filtration chromatography were dialyzed against 0.1 M phosphate buffer (pH 6.0) containing 0.9% NaCl and kept in the same buffer at various temperatures (60–70°C) for 5 min. The remaining LDH activity was assayed under the standard condition.21
RESULTS AND DISCUSSION Effect of NaCl concentration on coagulation temperature The coagulation method to determine EPT of food products is based upon the extractability of muscle proteins in water or salt solutions as a result of thin coagulation during heating.6 Table 1 shows the effect of NaCl concentration for extraction on the coagulation temperature. The samples were found to give an advanced coagulum with the extraction by 0.4 or 0.9% NaCl solution rather than by distilled water or 1.5% NaCl solution. Proteins extracted with distilled water produced a comparable coagulum up to 60°C, and also a considerable coagulum in 65°C- but not 70°C-heated samples. These findings are in support of those reported by Doesburg and Papendrof,23 and Coretti.6 For the filtrates obtained with distilled water and 0.4% NaCl solution, the relation of preheating temperature to coagulation temperature was rather poor when the preheated temperature of the former was higher than 60°C and that of the latter higher than 65°C. This might be due to an insufficient concentration of extracted proteins which could produce a coagulum. However, in the filtrates of
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Table 1 Coagulation temperatures of proteins extracted with different concentrations of NaCl from blue marlin block meats which were heated to different temperatures without holding time NaCl concentration used (%) 50.0°C 0 0.4 0.9 1.5 1 2
49.6–50.1 47.8–48.2 48.1–48.3 44.3–44.6
Minimum and maximum coagulation temperature after heat treatment 55.0°C 60.0°C 65.0°C 70.0°C 55.2–55.5 53.0–53.2 53.1–53.5 48.0–48.1
61.2–61.4 60.6–60.9 59.8–60.2 60.1–60.3
66.6–66.8 64.8–65.3 64.8–65.3 63.0–63.2
No coagulation1 72.3–72.4 71.2–72.0 Scattered2
No coagulum was observed up to 80°C. Scattered coagula were observed over 72°C.
Table 2 Coagulation temperature of proteins extracted from blue marlin meats which were heated at different temperatures with different holding times Sample Blue marlin (block meat)
Holding time (min)
50.0°C
0 5 15 30
48.1–48.3 48.9–49.0 49.6–50.0 50.2–50.4
Minimum and maximum coagulation temperature after heat treatment 55.0°C 60.0°C 65.0°C 67.0°C 68.0°C 70.0°C 53.1–53.5 53.6–53.9 53.9–54.4 55.0–55.5
extracts with 0.9% NaCl, comparable coagula were formed in the sample heated up to 70°C. The filtrates obtained with 1.5% NaCl extraction from 50°C-preheated samples became milky at 44.3°C, and produced an early coagulum in all the samples which were heated up to 65°C. However, only scattered coagula were observed at above 72°C in the samples heated at 70°C. The phenomenon of such results is not well understood at this time, but they are agreeable with the results of Visacki et al.24 in meat products. The irregular coagula from 1.5% NaCl solution suggested that this concentration might not be suitable for detecting EPT to which the product was processed. From these results, 0.9% NaCl solution was chosen to extract proteins from blue marlin meat for the remainder of this study, since the coagulation temperatures of 0.9% NaCl extract were well coincided with actual EPT.
Effects of sample preparation methods, holding times, and protein content on coagulation temperature The coagulation temperatures for the filtrates prepared with 0.9% NaCl solution from the samples of 5, 15, and 30 min holding times at 50, 55, 60, 65, and 67°C corresponded fairly well with the actual pre-heat treatments (Table 2). In the case of samples heated at 70°C, a considerable amount of coagula was formed in the 0 min holding time filtrate. However, after 5, 15 and 30 min holding times, a coagulum was formed only above 72°C. The best correspondence between EPT and coagulation temperature of the filtrates was observed in the samples
59.8–60.2 60.2–60.6 60.9–61.3 61.4–61.9
64.8–65.3 65.8–66.0 66.0–66.0 66.3–66.6
66.8–67.2 66.8–67.1 67.4–67.8 68.3–68.5
68.8–69.2 69.5–69.8 70.1–70.6 70.5–70.9
71.2–72.0 72.6–73.3 73.3–73.6 73.8–74.1
heated at 60°C and 65°C. Doesburg and Papendrof reported a similar correlation in hake Merluccius capensi, and also found coagula after heating to 70 and 80°C, but there was no close relationship observed between heating temperature and coagulation temperature.23 Lyon,25 Popescu and Din26 suggested that sample preparation methods which changed the size of particles, as well as time and temperature of protein extraction might affect the coagulation temperature. However, they did not include any data in their reports to support their suggestion. In the present study on heated blue marlin, we found that the sample preparation methods (block meat, ground meat, and homogenized meat) and holding times (5, 15, and 30 min) had no significant effect on the coagulation temperature (data not shown). These results are in agreement with those of Townsend et al.,5,27 Townsend and Blankenship3 and Doesburg and Papendrof.28 Because factors such as pH and ionic strength of NaCl extraction solutions, time and temperature of protein extraction, quality of products, or the presence of sugar compounds in products which may affect the coagulation temperature of filtrates, have not been thoroughly investigated, these should be the subject of further investigation. Moreover, the method which can estimate EPT above 68°C has to be developed, since the coagulation method could not determine higher EPT. We are planning to use a spectrophotometer with temperature controller and programmer for the determination of coagulation temperatures because the method used in the present study was subjective and lacking in accuracy. The biuret method was used in the present study to determine the relationship between protein concentra-
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tion of the filtrate and the coagulation temperature. The content of the soluble proteins extracted with 0.9% saline solution gradually decreased with increasing EPT and the remarkable decrease occurred between 50 and 60°C (data not shown). The content of the soluble proteins became low when EPT of the heated samples reached 60°C, because the major part of the soluble proteins had already been denatured between 50 and 60°C. These results are in agreement with those reported by Parsons and Patterson.29 When the protein content of the filtrate was below 5 mg/mL, it was not possible to detect EPT by the coagulation method. In all the cases, when the protein content of the filtrate was lower, the coagulation temperature of the filtrate was higher, indicating that there is an inverse relationship between soluble protein content of the filtrates and EPT. Comparable observations were also reported in meat products by Townsend et al.,5,27 Popescu and Din,26 and Davis and Anderson.30
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Table 3 Lactate dehydrogenase (LDH) and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) activity in filtrates prepared from blue marlin meat heated to various end-point temperatures with different holding times Temperature (°C)
Holding time (min)
LDH activity units/g meat
GAPDH activity units/g meat
Raw 65 65
– 0 30
568 675 586
498 43 39
67 67 67
0 15 30
282 185 98
17 8 0
68 68 68
0 5 15
47 42 31
0 0 0
69 69
0 30
26 20
0 0
70 70 70
0 15 30
12 8 4
0 0 0
72
0
0
0
An advantage of using SDS-PAGE is that it enables constant electrophoretic mobilities of proteins, independent
Fig. 1 Sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of 0.9% NaCl filtrates from blue marlin meats heated at different temperatures with different holding times. M, molecular weight marker; N, meat without heating.
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of isoelectric point and amino acid composition, which can be slightly modified during cooking.10 Figure 1 illustrates SDS-PAGE patterns of the proteins extracted with 0.9% NaCl solution from heated blue marlin meats which were heated at different temperatures with different holding times. Analysis of the raw meat revealed approximately 10 protein bands with molecular weights calculated between 23 and 87 kDa. Most protein bands observed in the raw meat filtrate were well detected when the meat was heated up to 50°C, but gradually disappeared with increasing heating temperature. These results are similar to those of Morioka and Shimizu31 in different fish species. The intensity of the band with a molecular weight of around 31 kDa did not change at 65°C during 30 min holding time and gradually decreased with increasing holding time at 67°C. This protein band disappeared at 68°C when the holding time was 15 min, and no protein bands were detected at 69°C (data not shown). These findings support the results reported by Huang et al.32 in fish proteins from frozen fish mince wash water. It is interesting to note that SDSPAGE patterns of the filtrates from heated blue marlin meats were closely related to the results of the coagulation method (Table 2). Therefore, it is evident that SDSPAGE could detect EPT of heated blue marlin meats up to 67°C with up to 30 min holding time. The protein with a molecular weight of about 31 kDa might be responsible for the formation of coagula at 67°C. The results described above have the following significances: (i) the disappearance of all the protein bands detected by the present electrophoretic method indicates that the blue marlin meat blocks were heated above 67°C; (ii) high degree of accuracy plus reproducibility using SDSPAGE confirm that the coagulation method used in this study offers a proper means to accurately determine EPT.
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Fig. 2 Gel filtration profiles of 0.9% NaCl filtrates from blue marlin meats heated at 65°C for 30 min. Column: Sephadex G-100 (2.8 ¥ 65 cm), elution buffer: 0.066 M carbonate buffer (pH 10) containing 0.2 M NaCl, flow rate: 0.4 mL/min. , absorbance at 280 nm; , relative lactate dehydrogenase activity. Void volume (Vo) and elution times of markers (a, 160 kDa; b, 67 kDa; c, 45 kDa) are indicated by arrows.
Identification and characterization of 31 kDa component It was revealed by SDS-PAGE analysis that the protein component with a molecular weight of about 31 kDa, thermally stable protein in blue marlin meat, might be responsible for the formation of coagulum above 65°C in the coagulation method. Therefore, the identification of a 31 kDa component was conducted in the following experiments. Based on the studies by Markert and Faulhaber,20 Pesce et al.33 Taniguchi et al.34 Nakagawa et al.35 and Hayashi et al.36 it was contemplated that this 31 kDa component may be either GAPDH or LDH subunit. However, GAPDH activity was not detected in the 0.9% NaCl filtrate prepared from blue marlin meat which had been heated at 67°C for 30 min (Table 3). Therefore, LDH activity of the filtrates from heated blue marlin meats was determined and the results are also presented in Table 3. Lactate dehydrogenase activity of raw blue marlin meat was 568 units/g and retained up to the heat
Fig. 3 Native polyacrylamide gel electrophoresis patterns of 0.9% NaCl filtrates prepared from blue marlin meats heated at 65°C for 30 min. (a) Stained by Coomassie brilliant blue; (b) stained by lactate dehydrogenase activity.
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treatment at 65°C for 30 min. The marked decline of LDH activity was observed when the meat was heated from 65 to 67°C, indicating that the enzyme activity profile is closely correlated to SDS-PAGE pattern as shown in Fig. 1. The activity was reduced to almost undetectable levels in the filtrates of 70°C-heated samples. These findings are in good agreement with the findings of Hayashi et al.36 From these results, it is suggested that 31 kDa component responsible for the coagulum formation above 65°C in the coagulation test is LDH subunit. In order to further elucidate the identification of 31 kDa component, Sephadex G-100 gel filtration chromatography of the filtrate prepared from blue marlin meat heated at 65°C for 30 min was conducted (Fig. 2). Since the sample had already been heat treated at 65°C for 30 min, most sarcoplasmic proteins were denatured and only two peaks were observed on the gel filtration chromatogram. Lactate dehydrogenase activity was detected in the second peak. Fractions 27–29 (Fig. 2) were pooled, dialyzed, freeze-dried, and used for further experiments. The molecular weight of the protein in fraction 28 was esti-
Fig. 4 Protein bands on sodium dodecylsulfate-polyacrylamide gel electrophoresis gels stained with Coomassie brilliant blue. (a) Stained protein previously separated by native polyacrylamide gel electrophoresis gels; (b) freeze-dried fraction (27–29) obtained by Sephadex G-100 gel filtration; (c) molecular weight markers.
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mated to be approximately 130 kDa from the elution pattern of molecular weight markers. The freeze-dried fraction (27–29) was applied on 7.5–15% native PAGE (Fig. 3). Protein bands were stained with Coomassie brilliant blue (Fig. 3a), and LDH was visualized by activity staining (Fig. 3b). It is obvious from Fig. 3 that there was a single protein band detected, which also possessed LDH activity. Then, the Coomassie brilliant bluestained protein band was cut out from the native PAGE gel and re-run on SDS-PAGE (Fig. 4a). There was a single protein band detected by the staining with Coomassie brilliant blue after electrophoresis and its mobility coincided with that of freeze-dried fraction (27–29) obtained from the gel filtration chromatography (Fig. 4b). The molecular weight of this protein band was calculated to be around 31 kDa. From these results, it is concluded the 31 kDa component present in the filtrate prepared from blue marlin meat heated at 65°C for 30 min is a subunit of LDH (EC 1. 1. 1. 27), since LDH is known to have a tetrametric quaternary structure.37,38 In the last experiment, thermal stability of LDH (fractions 27–29) obtained by Sephadex G-100 gel filtration was determined. Lactate dehydrogenase solution (1 mL) was heated at temperatures ranging 60–70°C, cooled in icewater after heating, and residual LDH activity was measured (Fig. 5). As shown in Fig. 5, the enzyme retained full activity at temperature up to 61°C, but the activity decreased gradually with increasing heating temperature. Lactate dehydrogenase lost its activity completely at 68°C. This phenomenon is fairly similar to the results given in Table 3. Therefore it is concluded that EPT of heated blue marlin meats can be also determined by measuring LDH activity of their filtrates.
Fig. 5 Heat inactivation of lactate dehydrogenase obtained by Sephadex G-100 gel filtration chromatography (fraction 27–29). Enzymes were heated for 5 min at the designated temperatures.
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ACKNOWLEDGMENT The authors would like to express their sincere thanks to Mr. Kazushige Usui of Kanagawa Prefectural Fisheries Research Institute for supplying frozen blue marlin meats.
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