Original Paper Objective Quality Evaluation of Commercial Spicy

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Original paper

Objective Quality Evaluation of Commercial Spicy Pollack Roe Products in Terms of Mechanical and Biochemical Properties

1 1 1 2 1* Chaoping Chen , Emiko Okazaki , Toru Suzuki , Ha Thi Nhu Nguyen and Kazufumi Osako

1Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan 2College of Aquaculture and Fisheries, Can Tho University, Can Tho, 92000, Vietnam

Received October 27, 2015 ; Accepted January 11, 2016

In order to identify objective parameters for quality evaluation, physical dimensions, pH, color parameters, proximate composition, water activity, lipid oxidation, free amino acids, mechanical properties, protein composition and differential scanning calorimetry of commercial Japanese spicy pollack roe products of differing quality were analyzed. Low-priced roe products showed significantly higher ( p < 0.05) average thiobarbituric acid reactive substances content than high-priced roe products from the same company. More bitter amino acids were detected in the low-priced, compared to the high-priced roe products from the same company. Ovary membrane and eggshell proteins in low-priced roe products were comprised of a relatively greater amount of low-molecular-weight components. Compared to low-priced roe products, high-priced ones showed higher values for the mechanical properties and thermal transition enthalpy of fish egg.

Keywords: spicy pollack roe products, mechanical properties, protein composition, free amino acids, differential scanning calorimetry

Introduction quantities exported to Korea (Alaska Seafood Marketing Institute Alaska pollack (Theragra chalcogramma) is a semi-pelagic (ASMI), 2011)iii), where it has a long history of use as a food schooling fish widely distributed in the temperate and arctic waters material. Manufacturers of roe products in Japan have their own of the North Pacific Ocean. It is a member of the cod family and expertise in classifying raw Alaska pollack roe into gamuko, mako, supports one of the largest single-species fisheries in the world and mizuko by maturity stage. Usually, only mako is accepted by (Smith 1981). It is harvested by Japan, Canada, Korea, Russia, and manufacturers as a standard roe material for manufacturing spicy the USA, and its aggregate biomass was an estimated 18 million pollack roe product which is a famous delicacy known as karashi metric tons in the Gulf of Alaska, Bering Sea, and Sea of Okhotsk mentaiko in Japanese. in 2010-2012 (Seafish, 2013)i). Generally, prices for spicy pollack roe products fluctuate The roe obtained from Alaska pollack is commercially widely according to the quality of different manufacturers. important, since it occupies only 5% of the fish weight but However, determination of quality is based on the empirical contributes about 31% of the commercial value of pollack products judgment of manufacturers. In recent years, the average price of (Balaban et al., 2012). It is an important export product of the roe products has fallen in Japan (Teikoku Databank, Ltd., 2011)iv). USA, accounting for 14,966 metric tons of total fishery products The lack of an objective quality evaluation system may have exported in 2013 (National Marine Fisheries Service (NMFS)ii), negatively impacted consumer perceptions of roe products and is 2013). The majority of these are exported to Japan, with limited considered as one of the reasons for the downward pressure on the

*To whom correspondence should be addressed. E-mail: [email protected] 338 C. Chen et al. average price of spicy pollack roe products. Therefore, it is calculating the protein content. Crude lipid was extracted according necessary to develop a clear standard for the quality evaluation of to the method of Folch et al. (1957). The crude carbohydrate roe products. content was obtained by subtracting the total composition ratios In this study, a total of 31 parameters were assessed in high- (%) of other chemical components from 100%. and low-priced roe products as follows: ovary physical dimensions Salt content, water phase salt content, and water activity The (ovary weight, ovary length, ovary condition factor, ovary same homogenate used for pH determination was used for salt membrane thickness, egg diameter), pH, color parameters (L*, a*, content measurement with a salt meter (Atago Co., Ltd., Tokyo, and b*), proximate composition (moisture, ash, lipid, protein, Japan). Water phase salt content was calculated from the values of carbohydrate), salt content, water phase salt content, peroxide salt content and moisture content (Cornu et al., 2006) as follows: value (PV), thiobarbituric acid reactive substances (TBARS), free Water phase salt content = Salt content / Moisture content amino acids (sweet amino acids, umami-tasting amino acids, and Water activity was measured by the method of Liu et al. (2013) bitter amino acids), mechanical properties (firmness, gumminess, with a Novasina water activity meter (model ms1; Novasina AG stickiness, tensile strength, and breaking strength), protein Ltd., Lachen, Switzerland) at 25.0 ± 0.3℃. composition (ovary membrane and eggshell), and differential PV and TBARS The PV and TBARS of crude lipid were scanning calorimetry (denaturation temperature and thermal determined according to the method described by Weng et al. transition enthalpy) in order to provide basic information for the (2009). establishment of evaluation standards for spicy pollack roe Free amino acids Free amino acid analysis was conducted products. according to the method of Kaneko et al. (2012) with a slight modification. To extract free amino acids, 10 g of roe sample was Materials and Methods homogenized in 10 mL deionized water; 10 mL of 10% Materials High-priced (about 1000 yen/100 g or about 8.3 trichloroacetic acid (TCA) was then added and the sample was dollars/100 g) and low-priced (about 400 yen/100 g or about 3.3 homogenized. The mixed solution was centrifuged at 10000 × g for dollars/100 g) spicy pollack roe products from 5 companies 15 min. The precipitate was washed with 10 mL 5% TCA to extract (marked A, B, C, D, and E) in Japan were sampled randomly. remaining free amino acids, and the same procedure was repeated Condition factor, egg diameter, and ovary membrane thickness 3 more times. The supernatant was filtered through filter paper (No. After measuring the weight and length of the ovary, the condition 5A) and its volume was adjusted to 100 mL. After filtering through factor was calculated by the method of Osako et al. (2003) using a cellulose membrane filter (0.45 μm; Toyo, Roshi Kaisha, Ltd., the following equation: Tokyo, Japan), its free amino acid composition was determined by an amino acid analysis system (Prominence; Shimadzu, Kyoto, Condition factor = 1000 × weight / (length) 3 ······Eq. 1 Japan) equipped with a column (Shim-pack Amino-Li, Egg diameter and ovary membrane thickness were measured 100 mm × 6.0 mm i.d.; column temperature, 39.0℃; Shimadzu) by a RHEONER II creep meter (RE2-33005B; Yamaden Co., Ltd., and pre-column (ShimpackISC-30/S0504 Li, 150 mm × 4.0 mm i.d. Tokyo, Japan) equipped with a cylindrical plunger (diameter = Shimadzu). A fluorescence detector (RF-10AXL, Shimadzu) was 8 mm), with a raising rate of the sample table at 1 mm/s. used for detecting amino acids. pH The determination of pH value was conducted according Mechanical properties Sample preparation for assessing to the method of Kung et al. (2008) with slight modifications. Each mechanical properties is shown in Figure 1. The firmness of roe sample (10 g) was homogenized in 90 mL deionized water using a products was assessed by measuring the strength at 20% strain homogenizer (DIAX600; Nikko Hansen & Co., Ltd., Japan), and using a RHEONER II creep meter (RE2-33005B; Yamaden Co., pH of the obtained homogenate was measured using a pH meter Ltd.) equipped with a cylindrical plunger (diameter = 16 mm). The (pH211; Hanna Instruments Co., Ltd., Japan). raising speed of the sample table was 1 mm/s. Color parameters The surface color tone of the roe products Ovary membranes were cut into a rectangular (1.5 cm × 3.0 cm) was measured by a color reader (CR-13; Konica Minolta Sensing film. A Tensipresser (TTP-50 BX II 2006; Taketomo Electric Inc., Inc., Tokyo, Japan), and was expressed as L* (lightness), a* Tokyo, Japan) was used to tear the rectangular film into two pieces. (redness / greenness), and b* (yellowness / blueness) values (CIE The tensile speed was set at 1 mm/s, and the maximum strength Laboratory System). Six ovaries were selected randomly for each was recorded as tensile strength. sample lot of roe product, and the surface color was measured at The food textural profile analysis developed by Friedman et al. four arbitrary points for each sample. (1963), Szczesniak et al. (1963), and Bourne (1978) was used to Proximate composition Moisture and crude ash contents were assess the gumminess and stickiness of ovary round slice. The analyzed according to the methods of AOAC (1999). Total nitrogen ovary round slice (height = 15 mm, diameter > 16 mm) was pressed content was determined by the Kjeldahl method (Bradstreet et al., twice on its center by the RHEONER II creep meter equipped with 1954) and a nitrogen conversion factor of 6.25 was used for a cylindrical plunger (diameter = 16 mm). The plunger penetration Objective Quality Evaluation of mentaiko 339

Fig. 2. Typical force-time curve of the ovary round slice of spicy pollack roe products. Fig. 1. Preparation of samples for mechanical properties measurement.

was 20% and the raising speed of the sample table was 1 mm/s. modifications. Fish eggs from each company were taken from six The force-time curve is shown in Figure 2, and consists of 2 roe products with the same grade randomly and mixed. The mixed positive peaks and 2 negative peaks. According to Bourne (1978), sample (2.0 g) was homogenized in 6 mL pre-cooled 20 mM Tris- the peak force during the first bite is defined as hardness, the ratio HCl buffer (pH = 8.0) containing 10 mM EDTA and 2 mM DTT, of the positive force area during the second bite to that during the and then centrifuged at 45,000 × g for 30 min at 4℃. The obtained first bite (Area 2 / Area 1) is defined as cohesiveness, “hardness × pellet was washed by repeating the procedure 3 more times to cohesiveness” is defined as gumminess, and stickiness remove any remaining soluble protein. The washed pellet was (adhesiveness) is defined as the negative force area for the first bite collected as eggshell protein. The eggshell protein was (Area 3). homogenized in 5 mL SDS solution (2% SDS, 8 M urea, 2% The breaking strength of fish egg was measured by the 2-mercaptoethanol in 20 mM Tris-HCl, pH = 8.0), and heated in RHEONER II creep meter equipped with a cylindrical plunger boiling water for 2 min. After cooling, the homogenate was stirred (diameter = 8 mm). The raising speed of the sample table was at room temperature for 12 h, and then centrifuged at 10,000 × g 1 mm/s. Each measurement was conducted with 18 repeats, and the for 20 min. The obtained supernatant was collected for SDS-PAGE top 5 values and the bottom 5 values were deleted. of eggshell protein. Sample preparation for SDS-PAGE of ovary membrane protein The eggshell protein was not completely solubilized in SDS and its solubility Ovary membrane materials were taken from six solution. The insoluble eggshell protein was visible and its roe products with the same grade randomly, and were then chopped solubility could not be measured by the same method used for and mixed. The mixed sample (0.5 g) was homogenized in 5 mL measuring the solubility of ovary membrane protein. However, SDS solution (2% SDS, 8 M urea, 2% 2-mercaptoethanol in 20 mM eggshell protein was completely solubilized in 2 N NaOH as Tris-HCl, pH = 8.0) in a centrifuge tube and heated in boiling water reported by Iuchi et al. (1991). Therefore, eggshell protein for 2 min. After cooling, the homogenate was stirred at room solubility in SDS solution was conducted according to the method temperature for 12 h, and then centrifuged at 10,000 × g for 20 min. of Benjakul and Visessanguan (2003) with slight modifications. The supernatant thus obtained was used for SDS-PAGE of ovary TCA solution was added to the homogenate or its supernatant to a membrane proteins. The protein concentration was measured by final concentration of 10% to precipitate protein in the SDS the method of Lowry et al. (1951). The ovary membrane solubility solution. The mixed solution was centrifuged at 10,000 × g for in SDS solution was measured according to the method of Chen et 30 min. The obtained pellet was washed with 10% TCA 3 times, al. (2012). and then solubilized in 2 N NaOH. The protein concentration in Sample preparation for SDS-PAGE of eggshell protein and its 2 N NaOH was measured by the method of Lowry et al. (1951). solubility Preparation of eggshell protein for SDS-PAGE was Protein solubility in the SDS solution was calculated by the ratio of according to the method of Bekhit et al. (2009) with slight the amount of protein solubilized in the supernatant and total 340 C. Chen et al. protein in the homogenate. priced products. This is most likely due to companies B and C SDS-PAGE SDS-PAGE was performed according to the using ovaries of a low maturity degree to produce low-priced roe method of Laemmli et al. (1970) using a 15% polyacrylamide gel products, since the diameter of cod eggs increases with the degree (AE-6000, NPU-7.5L PAGEL; Atto Co., Tokyo, Japan). Each of maturity (Katsiadaki et al., 1999). As maturity is closely related sample (10 µg) was applied to the polyacrylamide gel and subjected to the taste of roe products (Uchiumi et al., 2009a), egg diameter, to electrophoresis at a constant current of 20 mA using a compact- an indicator of ovary maturity, may be applicable as one of the PAGE apparatus (Atto Co.). The polyacrylamide gel was stained quality parameters. with 0.05% Coomassie Brilliant Blue R-250 in methanol/acetic The pH of the commercial roe products examined ranged from acid/water (5:10:85% v/v), and then destained with methanol/acetic 5.75 to 6.17 (Table 1). Roe products from the same company acid/water (30:10:60% v/v). A protein ladder (Thermo Fisher showed no significant differences in pH, but pH values differed Scientific Inc., Massachusetts, USA) with molecular weights between companies. This is most likely due to individual ranging from 10 to 200 kDa was used. companies adjusting the pH values of roe products according to Differential scanning calorimetry The measurements were their own processing standards. Moreover, no correlation was conducted with a Shimadzu DSC-50 following the method of found between the pH value and quality of roe products from the Schubring (2004) with slight modifications. Samples (30 – 50 mg same company. fish eggs or ovary membrane) were weighed (±0.1 mg) and sealed The surface color parameters of the roe products examined are in aluminum pans. Triplicate aluminum pans were heated from presented in Table 1. The L* values (lightness) of high-priced 10℃ to 145℃ at a scanning rate of 5℃/min with an empty products ranged from 36.5 – 44.5, and those of low-priced products aluminum pan as reference. Results were displayed as average were from 36.9 to 40.4. The a* values (redness) of high-priced curves in the figures. The transition temperature (Td, ℃) and products ranged from 6.2 to 24.8, and those of low-priced products transition enthalpy (ΔH, mJ/mg) were estimated from the peak of were from 6.6 to 34.4. The b* (yellowness) values of high-priced the DSC transition curve. products ranged from 6.2 to 13.6, while those of low-priced Statistical analysis Data analysis was conducted using products were from 9.9 to 15.9. According to Ueda et al. (2009), Microsoft Excel Statistical Analysis 2012 (Social Survey Research the optimum values of L*, a*, and b* of cod roe for consumer Information Co., Ltd., Tokyo, Japan). Results were compared preference are 45.22, 8.68, and 9.15, respectively. The average based on the multiple comparison test of Tukey–Kramer. In all color values of all examined high-priced products (L* = 40.3, a* = cases, the criterion for statistical significance was set atp < 0.05. 12.0, and b* = 9.8) were closer to the reported optimum values than those of low-priced roe products (L* = 39.0, a* = 15.3, and b* Results and Discussion = 11.9). Processing of spicy pollack roe products can lead to Ovary physical dimensions, pH, and color parameters Ovary changes in the proximate composition and structure of roe physical dimensions of commercial spicy pollack roe samples are (Hayabuchi et al., 1997), which may consequently affect the color presented in Table 1. The ovary weight of high-priced spicy of the final products. According to our interviews with roe product pollack roe products ranged from 33.3 g to 96.2 g, comparable to manufacturers, pigments or color-fixing agents, such as sodium that of the low-priced products (33.6 – 54.1 g). Companies B, C, nitrite, are sometimes used to adjust the color tone of the final roe and E provided high-priced roe products with higher weight values products. Therefore, the color tone of roe products may not be a than those of low-priced products. Only the products from suitable parameter for quality evaluation. company C showed significant differences ( p < 0.05) in ovary Proximate composition Few significant differences in weight and length values between high- and low-priced products. proximate composition between high- and low-priced roe products Ovary weight and length are not only affected by maturity stage, were observed. Respective average values of moisture, protein, but also by fish age (Oosthuizen and Daan, 1974; Katsiadaki et al., ash, lipid, and carbohydrate contents of high-priced roe products 1999), which may explain why the weight and length values, were 68.1%, 22.6%, 5.6%, 3.3%, and 0.4%, and those of low- especially ovary weight values, showed large variability. The ovary priced products were 68.2%, 22.4%, 5.6%, 3.4%, and 0.4% (Table membrane thickness value (about 0.18 mm) did not differ between 2). Although the moisture content of roe material is dependent on high- and low-priced products, regardless of the producer. the maturity stage (Uchiumi et al., 2009a), the proximate Therefore, measurements of ovary weight, ovary length, condition composition of processed roe, especially moisture content, changes factor, and thickness of the ovary membrane may not be applicable during manufacturing (Hayabuchi et al., 1997). Therefore, the parameters for the objective quality evaluation of roe products. quality of processed roe may be difficult to evaluate using The egg diameter of high-priced products occupied a narrow proximate composition parameters. range (0.53 – 0.61 mm) compared to the low-priced products Salt content, water phase salt content, and water activity Salt (0.34 – 0.51 mm). The average egg diameter of high-priced roe content and water phase salt content, and water activity of roe products from all 5 companies was higher than that of the low- products are presented in Table 2. The average salt content and Objective Quality Evaluation of mentaiko 341

Table 1. Physical dimensions, pH, and color parameters of spicy pollack roe products.

A B C H L H L H L Ovary weight 33.3 ± 2.0c 33.6 ± 1.7c 52.7 ± 6.7bc 46.1 ± 2.0bc 96.2 ± 8.9a 40.0 ± 1.7c (g, n=6) Ovary length 9.5 ± 0.8c 9.9 ± 0.7bc 11.0 ± 0.6b 10.6 ± 0.6bc 12.5 ± 0.8a 10.6 ± 0.4bc (cm, n=6) Condition factor 40.1 ± 9.5b 35.1 ± 7.6b 39.9 ± 5.1b 39.0 ± 5.8b 50.9 ± 11.1ab 34.1 ± 3.8b (kg/cm3, n=6) Egg diameter 0.53 ± 0.06ab 0.51 ± 0.05b 0.53 ± 0.05ab 0.46 ± 0.07b 0.61 ± 0.05a 0.34 ± 0.08c (mm, n=8) Ovary membrane 0.17 ± 0.01a 0.16 ± 0.02a 0.18 ± 0.05a 0.18 ± 0.03a 0.19 ± 0.03a 0.20 ± 0.04a thickness (mm, n=6) pH (n=4) 5.98 ± 0.08ab 5.95 ± 0.03b 6.17 ± 0.03a 6.15 ± 0.04ab 5.77 ± 0.01b 5.75 ± 0.01b Color parameters L* (n=6) 44.5 ± 3.8a 36.9 ± 1.8c 41.3 ± 0.9bc 40.4 ± 2.0bc 37.7 ± 3.3bc 40.0 ± 2.2bc a* (n=6) 11.6 ± 3.3cd 17.1 ± 2.4bc 6.4 ± 1.2d 6.6 ± 1.3d 11.1 ± 0.6cd 11.6 ± 2.2cd b* (n=6) 13.6 ± 2.6ab 13.2 ± 2.2ab 7.4 ± 0.6b 10.1 ± 1.4b 7.6 ± 1.7b 10.8 ± 0.9b D E All examined companies H L H L H L Ovary weight 55.9 ± 2.6b 54.1 ± 7.5bc 50.9 ± 4.9bc 45.9 ± 1.2bc 57.8 ± 23.2b 43.9 ± 7.6bc (g, n=6) Ovary length 10.1 ± 0.7bc 9.9 ± 0.4bc 11.1 ± 0.7b 11.0 ± 0.6b 10.8 ± 1.2bc 10.4 ± 0.5bc (cm, n=6) Condition factor 56.6 ± 12.8a 56.6 ± 9.3a 38.1 ± 6.5b 35.4 ± 5.3b 44.1 ± 8.2ab 40.1 ± 9.5ab (kg/cm3, n=6) Egg diameter 0.54 ± 0.05ab 0.49 ± 0.0b 0.53 ± 0.05ab 0.50 ± 0.06b 0.55 ± 0.04ab 0.46 ± 0.07b (mm, n=8) Ovary membrane 0.19 ± 0.03a 0.18 ± 0.03a 0.19 ± 0.04a 0.18 ± 0.03a 0.18 ± 0.01a 0.18 ± 0.01a thickness (mm, n=6) pH (n=4) 6.12 ± 0.12ab 6.06 ± 0.04ab 6.08 ± 0.03ab 6.08 ± 0.02ab 6.02 ± 0.16ab 6.00 ± 0.15ab Color parameters L* (n=6) 36.5 ± 1.2c 37.8 ± 1.8bc 41.6 ± 1.9b 39.7 ± 1.1bc 40.3 ± 3.2bc 39.0 ± 1.5bc a* (n=6) 24.8 ± 3.2b 34.4 ± 2.1a 6.2 ± 0.7d 6.7 ± 0.8d 12.0 ± 7.6cd 15.3 ± 11.5c b* (n=6) 13.5 ± 2.8ab 15.9 ± 2.1a 6.2 ± 0.8b 9.9 ± 1.4b 9.8 ± 3.4b 11.9 ± 2.6ab

The spicy pollack roe products from 5 companies are marked as A, B, C, D, and E. “H” and “L” denote high-level and low-level roe products, respectively. Different superscripts in the same row indicate statistical differences ( p < 0.05). Data are expressed as mean ± standard deviation. water phase salt content of the high-priced roe products were 4.6% al., 2009a). and 6.8%, while those of low-priced products were 5.2% and 7.5%. The average water activity values of high- and low-priced roe Companies C and E provided low-priced roe products containing products were 0.94 and 0.93, respectively, not significantly significantly higher salt content ( p < 0.05) than the high-priced different. Roe products from company D and low-priced roe products. It is possible that the roe material of the low-priced products from companies C and E showed low water activity products suffered greater damage from longer frozen storage than (0.92), since they showed comparative high water phase salt the high-priced products, resulting in increased NaCl absorbance content compared to other roe products. during salting. Another possible reason for the high salt content of PV and TBARS The crude lipid extracted from roe products the low-priced roe products, especially those from company C, is was analyzed for PV and TBARS (Table 2). Both PV and TBARS an attempt to reduce the high moisture content of roe material with of low-priced roe products (13.1 _ 24.8 meq/kg for PV, and a low maturity degree, since it was reported that roe material with a 67.3 _ 176.8 mg MDA/kg for TBARS) were higher than those of low maturity degree contained high moisture content (Uchiumi et high-priced roe products (8.8 _ 13.6 meq/kg for PV, and 342 C. Chen et al.

Table 2. Proximate composition, lipid oxidation, and water activity of spicy pollack roe products.

A B C H L H L H L Moisture content 67.6 ± 2.0a 69.0 ± 2.4a 67.1 ± 1.4a 68.2 ± 2.1a 70.0 ± 1.1a 69.5 ± 1.7a (%) Protein content 24.0 ± 1.6a 22.8 ± 2.0ab 23.7 ± 1.5ab 23.2 ± 1.5ab 20.2 ± 1.0b 20.5 ± 0.7ab (%) Ash content 5.1 ± 0.1a 5.1 ± 0.2a 5.5 ± 0.8a 5.2 ± 0.6a 5.9 ± 1.1a 6.6 ± 0.6a (%) Lipid content 2.8 ± 0.6a 2.9 ± 0.5a 3.2 ± 0.6a 3.0 ± 0.7a 3.5 ± 0.2a 3.1 ± 1.3a (%) Carbohydrate content 0.5 ± 0.1a 0.3 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.2a (%) Salt content 4.5 ± 0.0bc 4.5 ± 0.0bc 4.5 ± 0.1bc 4.4 ± 0.1c 4.3 ± 0.1c 6.0 ± 0.1a (g/100g) Water phase salt content 6.6 ± 0.1bc 6.5 ± 0.2bc 6.7 ± 0.1bc 6.5 ± 0.3bc 6.2 ± 0.0c 8.7 ± 0.3a (g/100ml) Water activity 0.94 ± 0.00b 0.94 ± 0.00b 0.94 ± 0.00b 0.94 ± 0.00b 0.95 ± 0.01a 0.92 ± 0.00c

PV 13.6 ± 1.5b 17.2 ± 0.6ab 8.8 ± 1.5b 23.5 ± 5.8a 13.2 ± 4.6b 19.7 ± 0.7ab (meq/kg) TBARS 71.2 ± 5.3c 95.1 ± 9.0bc 53.9 ± 9.8c 151.4 ± 7.7ab 67.5 ± 10.8c 99.9 ± 7.2bc (mg MDA/kg) D E All examined companies H L H L H L Moisture content 68.0 ± 1.2a 68.3 ± 0.7a 67.7 ± 0.9a 66.3 ± 2.8a 68.1 ± 1.1a 68.2 ± 1.2a (%) Protein content 22.4 ± 1.1ab 21.8 ± 1.2ab 22.7 ± 1.4ab 23.8 ± 2.5ab 22.6 ± 1.5ab 22.4 ± 1.3ab (%) Ash content 5.8 ± 0.7a 5.7 ± 0.9a 5.8 ± 1.5a 5.6 ± 0.9a 5.6 ± 0.3a 5.6 ± 0.6a (%) Lipid content 3.5 ± 0.3a 3.9 ± 0.3a 3.5 ± 0.4a 4.0 ± 0.3a 3.3 ± 0.3a 3.4 ± 0.5a (%) Carbohydrate content 0.3 ± 0.2a 0.3 ± 0.1a 0.3 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.0a (%) Salt content 5.5 ± 0.1ab 5.5 ± 0.3ab 4.4 ± 0.2c 5.3 ± 0.1ab 4.6 ± 0.5bc 5.2 ± 0.7b (g/100g) Water phase salt 8.1 ± 0.1ab 8.1 ± 0.4ab 6.4 ± 0.3bc 8.0 ± 0.3ab 6.8 ± 0.8bc 7.5 ± 1.0b content (g/100ml) Water activity 0.92 ± 0.01bc 0. 92 ± 0. 01bc 0.94 ± 0.00b 0.92 ± 0.00c 0.94 ± 0.01b 0. 93 ± 0. 01bc

PV 13.6 ± 1.5b 24.8 ± 8.4a 9.0 ± 0.6b 13.1 ± 1.4b 11.6 ± 2.5b 19.7 ± 4.7ab (meq/kg) TBARS 75.5 ± 3.2c 176.8 ± 12.8a 54.9 ± 1.5c 67.3 ± 1.9c 64.6 ± 9.7c 118.1 ± 44.7b (mg MDA/kg)

Abbreviations are the same as given in Table 1. Different superscripts in the same row indicate statistical differences ( p < 0.05). Data are expressed as mean ± standard deviation (n = 4). Objective Quality Evaluation of mentaiko 343

Table 3. Free amino acids in spicy pollack roe products.

Amino acid A B C (mg/100g) Taste H L H L H L Taurine - 87.1 ± 9.8bc 96.2 ± 6.1bc 66.4 ± 8.4c 76.0 ± 11.1bc 110.7 ± 9.1b 182.3 ± 10.4a Aspartic acid U 45.3 ± 4.2b 54.1 ± 1.4b 53.7 ± 2.0b 71.4 ± 2.8b 76.6 ± 2.1b 119.6 ± 2.7b Threonine S 26.3 ± 2.4c 33.6 ± 1.5bc 24.0 ± 1.7c 30.3 ± 2.4c 33.7 ± 1.8bc 58.8 ± 2.3b Serine S 38.1 ± 1.6c 48.6 ± 1.1bc 43.4 ± 1.5c 54.2 ± 2.0bc 65.1 ± 1.6bc 123.2 ± 1.9ab Glutamic acid U 1928.1 ± 252.4ab 1134.8 ± 153.0b 2223.6 ± 210.3ab 2253.7 ± 266.6ab 2430.9 ± 230.3a 2322.9 ± 251.4ab Proline S 27.0 ± 3.0ab 31.3 ± 1.7ab 30.0 ± 2.2ab 36.0 ± 2.3ab 38.3 ± 2.6ab 45.3 ± 2.2a Glycine S 159.6 ± 38.9b 29.4 ± 1.4b 138.0 ± 16.7b 147.3 ± 18.3b 90.2 ± 22.3b 97.8 ± 58.2b Alanine S 45.8 ± 6.9c 52.9 ± 4.6c 112.3 ± 6.4bc 153.7 ± 9.1bc 860.3 ± 87.8ab 1210.1 ± 147.8a Valine B 34.0 ± 7.7c 45.4 ± 4.9bc 33.7 ± 6.8c 46.1 ± 9.3bc 46.6 ± 7.2bc 83.8 ± 8.8bc Methionine B 15.1 ± 1.6b 20.0 ± 0.9b 16.8 ± 1.2b 18.3 ± 1.2b 18.2 ± 1.4b 30.9 ± 1.2b Isoleucine B 23.4 ± 5.6b 32.5 ± 3.4b 20.5 ± 4.6b 29.4 ± 5.9b 27.0 ± 5.1b 50.1 ± 5.5b Leucine B 56.4 ± 5.8b 70.3 ± 3.0b 51.0 ± 4.0b 48.4 ± 4.6b 84.6 ± 4.6b 135.7 ± 4.4b Tyrosine B 28.0 ± 0.7c 39.7 ± 0.4c 25.8 ± 4.9c 43.1 ± 4.6c 46.8 ± 6.3bc 102.7 ± 4.5b Phenylalanine B 25.0 ± 3.3c 34.9 ± 1.7bc 21.0 ± 2.3c 28.9 ± 1.7bc 31.0 ± 2.8bc 52.1 ± 1.7b Histidine B 13.4 ± 1.2c 17.4 ± 0.7bc 12.6 ± 0.9c 13.6 ± 1.1c 18.5 ± 1.0bc 33.8 ± 1.1ab Lysine B 45.2 ± 1.1ab 60.1 ± 2.5ab 35.7 ± 6.9b 46.4 ± 7.01ab 41.1 ± 8.1ab 64.0 ± 6.8a Arginine B 47.1 ± 2.9c 59.8 ± 0.8bc 35.9 ± 3.4c 46.8 ± 3.0c 47.4 ± 4.1c 87.7 ± 2.9b Total FAA 2645.0 ± 229.5b 1860.9 ± 123.2c 2944.4 ± 170.7b 3143.4 ± 219.3b 4066.8 ± 113.9ab 4771.9 ± 76.9a Sweetness 296.9 ± 29.1bc 195.7 ± 6.4c 347.7 ± 17.2bc 421.4 ± 29.6bc 1087.5 ± 73.3b 1535.1 ± 109.3ab Umami 1973.3 ± 248.9ab 1188.9 ± 151.6b 2277.3 ± 208.3ab 2325.1 ± 263.7ab 2507.4 ± 228.3a 2442.5 ± 248.8ab Bitterness 287.7 ± 28.3c 380.1 ± 17.1bc 253.0 ± 32.9c 320.8 ± 36.8c 361.1 ± 39.0bc 640.8 ± 35.1ab D H All examined companies H L H L H L Taurine - 99.1 ± 4.6bc 106.3 ± 9.3bc 84.6 ± 8.9bc 97.7 ± 5.6bc 89.6 ± 16.6bc 111.7 ± 41.0b Aspartic acid U 253.1 ± 1.0ab 280.8 ± 2.1a 245.5 ± 2.1ab 221.5 ± 1.3ab 134.8 ± 105.2b 149.5 ± 98.1b Threonine S 73.5 ± 1.0ab 87.2 ± 1.8a 70.1 ± 1.8ab 68.9 ± 1.1ab 45.5 ± 24.3bc 55.8 ± 24.0bc Serine S 171.3 ± 0.8ab 193.4 ± 1.7a 170.4 ± 1.6ab 155.1 ± 1.0ab 97.7 ± 67.6bc 114.9 ± 63.1b Glutamic acid U 1580.2 ± 120.3b 1414.0 ± 237.5b 1451.4 ± 223.4b 1228.6 ± 141.0b 1922.8 ± 414.8ab 1670.8 ± 573.1b Proline S 0.0c 0.0c 0.0c 0.0c 19.1 ± 17.9b 22.5 ± 21.2b Glycine S 1116.7 ± 30.8a 931.0 ± 79.1ab 1070.8 ± 143.0a 845.1 ± 79.8ab 515.1 ± 529.1b 410.1 ± 439.3b Alanine S 564.9 ± 5.6b 581.6 ± 55.9b 531.7 ± 59.5b 492.9 ± 35.7bc 423.0 ± 339.9bc 498.2 ± 455.7bc Valine B 123.5 ± 3.5ab 141.7 ± 7.4a 115.4 ± 7.1ab 112.2 ± 4.4ab 70.6 ± 44.9bc 85.8 ± 41.9b Methionine B 88.7 ± 0.8a 93.7 ± 1.5a 84.8 ± 1.3ab 83.8 ± 0.9ab 44.7 ± 38.4b 49.3 ± 36.5b Isoleucine B 71.4 ± 2.6ab 88.1 ± 5.2a 72.6 ± 4.9ab 73.8 ± 3.1ab 43.0 ± 26.6b 54.8 ± 25.7b Leucine B 165.9 ± 2.6ab 196.0 ± 4.8a 170.6 ± 4.4ab 194.3 ± 2.8ab 105.7 ± 58.5b 128.9 ± 68.4b Tyrosine B 107.5 ± 3.9ab 155.8 ± 6.7a 110.5 ± 6.0ab 115.8 ± 4.0ab 63.7 ± 42.1bc 91.4 ± 49.7bc Phenylalanine B 63.0 ± 1.7ab 77.5 ± 3.0a 62.1 ± 2.6ab 64.0 ± 1.7ab 40.4 ± 20.5bc 51.4 ± 20.1b Histidine B 33.6 ± 0.6ab 42.2 ± 1.1a 34.6 ± 1.0ab 33.0 ± 0.6ab 22.5 ± 10.8bc 28.0 ± 12.1b Lysine B 15.7 ± 4.8b 15.0 ± 8.5b 13.4 ± 7.7b 17.7 ± 5.0b 30.2 ± 14.7b 40.6 ± 23.1ab Arginine B 100.4 ± 2.5ab 134.5 ± 4.3a 105.4 ± 3.8ab 89.4 ± 2.5b 67.2 ± 32.9bc 83.7 ± 33.8b Total FAA 4628.6 ± 101.1a 4556.9 ± 198.5a 4375.2 ± 186.4a 3893.9 ± 117.9ab 3732.0 ± 884.8ab 3645.4 ± 1183.0ab Sweetness 1926.5 ± 23.7a 1793.2 ± 57.4ab 1843.0 ± 86.5ab 1562.0 ± 42.6ab 1100.3 ± 782.0b 1101.5 ± 735.1b Umami 1833.4 ± 119.4ab 1694.8 ± 235.4b 1696.9 ± 221.4b 1450.2 ± 139.8b 2057.7 ± 330.9ab 1820.3 ± 546.2b Bitterness 769.6 ± 22.3ab 944.5 ± 40.8a 769.4 ± 37.2ab 784.0 ± 24.1ab 488.2 ± 259.8bc 614.0 ± 264.3b

Abbreviations are the same as given in Table 1. “Sweetness (S)”, “Umami (U)” and “Bitterness (B)” mean total amount of sweet amino acids (threonine, serine, glycine, and alanine), umami-tasting amino acids (aspartic acid and glutamic acid) and bitter amino acids (valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine), respectively. Different superscripts in the same row indicate statistical differences ( p < 0.05). Data are expressed as mean ± standard deviation (n = 4). 344 C. Chen et al.

53.9 _ 75.5 mg MDA/kg for TBARS) from the same company. A comparatively longer storage period may be the reason for the higher values of PV and TBARS of low-priced roe products compared to the high-priced roe products. Free amino acids The 17 typical free amino acids were detected and identified in spicy pollack roe products (Table 3). Sweet amino acids (glycine and alanine) and umami-tasting amino acids (glutamic acid and aspartic acid) were the main amino acids contained in the roe products. High-priced roe products showed slightly higher average contents of umami-tasting amino acids (2057.7 mg/100 g) than those (1820.3 mg/100 g) of low-priced roe products. However, umami-tasting amino acids, as well as sweet amino acids, may be derived from food additives (Ueda et al., 2009), thus they cannot be considered as parameters for the quality evaluation of spicy roe products. Bitter amino acids, including valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine are generally considered as endogenous amino acids in roe materials. Comparing within the same company, all 5 companies provided low-priced products containing higher contents (320.8 mg/100 g to 944.5 mg/100 g) of bitter amino acids than those contained in high- priced roe products (253.0 mg/100 g to 769.6 mg/100 g). Uchiumi et al. (2009b) demonstrated that the immature ovaries of Alaska pollack contained greater bitter amino acids content compared to mature ovaries. Therefore, the bitter amino acids content of spicy pollack roe was partly dependent on the maturity degree. Moreover, the low-priced products from companies B and C showed small egg diameters, indicating immature ovaries, and contained higher levels of bitter amino acids. It is also possible that a longer storage period may be another reason for the higher contents of bitter amino acids. Therefore, the content of bitter free amino acids is a potential parameter for the quality evaluation of roe products. Mechanical properties Firmness (hardness) is the force required to attain a given strain (or deformation) of a material (Szczesniak et al., 1963). As presented in Figure 3, except for roe products from company B, the ovary firmness values of high-priced _ Fig. 3. Mechanical properties of spicy pollack roe products. products (85 198 gw) were higher than those of low-priced Abbreviations are the same as given in Table 1. products (41 _ 99 gw). Based on interviews with the manufacturers of roe products, firmness is typically evaluated by touch, and a judgment is made by personal empirical experience. Roe products (21.1 _ 66.2 gw) were higher than those of low-priced roe products with higher ovary firmness are considered as superior. The current (11.7 _ 30.5 gw); in contrast, the stickiness values of ovary round method in this study for measuring the firmness of roe products can slices from high-priced products (8.2 _ 43.9 J/m3) were lower than be presented to manufacturers as an objective, convenient, and those of low-priced products (13.8 _ 50.1 J/m3). The gumminess of nondestructive mechanical method to evaluate the firmness of roe a sample is related to the energy required to disintegrate a semisolid products. All of the high-priced roe products showed higher tensile food to a state ready for swallowing, and the stickiness is strength values (69 _ 213 gw/cm2) of ovary membrane than those represented by the work necessary to pull the plunger away from of low-priced roe products (24 _ 128 gw/cm2). The stronger ovary the food sample (Szczesniak et al., 1963). membranes of the high-priced roe products may be considered as While most commercial roe products are prepared from frozen one of the reasons for their higher ovary firmness values compared roe material, those prepared from fresh roe are generally considered to low-priced roe products. Except for company B, the gumminess superior. Frozen storage causes physical damage to the ovaries, and values of ovary round slices of high-priced products improper freezing conditions due to slow freezing or extended Objective Quality Evaluation of mentaiko 345 frozen storage causes greater drip loss from ovaries during thawing (Uchiumi et al., 2009b). The relatively lower values of egg breaking strength of low-priced roe products reflect the higher degree of physical damage during frozen storage. Most drip loss from ovaries during thawing consists of yolk protein from broken fish eggs, which may increase product stickiness. Therefore, high stickiness values indicate a high ratio of broken fish eggs in the ovaries, which is considered one of the reasons for the decline in gumminess value. According to our communications with manufacturers of spicy pollack roe products, low-priced roe products are usually processed from roe materials subjected to an extended period of frozen storage. This may help explain why, within the same company, low-priced roe products showed higher values of stickiness and generally lower values of gumminess. Almost all high-priced roe products showed significantly greater values of egg breaking strength (18.1 _ 29.7 gw) than those of low-priced roe products (4.9 _ 20.0 gw). Egg breaking strength, which is mainly affected by processing method, is considered to be an important texture parameter that is closely related to a grainy feel, so called “tsubukan” in Japanese; and roe products with a stronger grainy feel are usually considered to be superior. The higher ovary firmness and gumminess values of high-priced roe products may also be attributed to the greater strength values of fish eggs. Protein composition and solubility in SDS solution SDS- Fig. 4. SDS-PAGE of ovary membrane protein (I) and eggshell PAGE patterns of ovary membrane protein composition are shown protein (II). Abbreviations are the same as given in Table 1. in Figure 4 (I). The SDS-PAGE patterns of ovary membrane protein differed between high- and low-priced products. Greater high-molecular-weight proteins (> 100 kDa) were observed in the solutions of different ionic composition from the ovary medium ovary membrane of high-priced products compared to low-priced (Lönning and Kjörsvik, 1984). Oppen-Berntsen et al. (1990) products, which was especially obvious in the roe products from demonstrated that nonactivated eggshell from cod primarily company B. The ovary membrane proteins from high-priced consists of only 3 protein monomers, α (74 kDa), β (54 kDa), and γ products showed less solubility in SDS solution (86% _ 91%) (47 kDa); and a transglutaminase-dependent eggshell protein compared to the low-priced products (91% _ 99%). Differences in crosslinking, accompanied by a decline in protein solubility, may the protein composition of ovary membranes between high- and be responsible for the egg hardening. During the processing of low-priced roe products may explain the differences in tensile spicy pollack roe products, protein crosslinking of the eggshell can strength values, as mentioned previously. be activated by soaking the eggs in a salt solution, and the amount SDS-PAGE analysis of eggshell protein is shown in Figure 4 of high-molecular-weight eggshell protein may reflect the degree (II). The major eggshell proteins from samples AH, AL, DH, and of protein crosslinking. The relatively higher ratio of high- EH showed a molecular weight of about 100 kDa, while the major molecular-weight proteins in the eggshell of high-priced roe proteins in BH and DL were about 50 kDa. Eggshell proteins products may be one reason for their generally higher ovary consisting of both 50 kDa and 100 kDa were mainly contained in firmness, gumminess, and egg breaking strength compared to low- CH, CL, and EL. The molecular weight of eggshell proteins in BL priced products from the same company. was especially low, about 50 kDa or less. In brief, the eggshell of Differential scanning calorimetry Thermal transition curve, high-priced products contained more high-molecular-weight denaturation temperature, and transition enthalpy of fish eggs are proteins, compared to the low-priced products within the same shown in Figure 5. Only one distinct peak was observed in the company. The solubility values of eggshell proteins in high-priced thermal transition curve for the roe products, likely attributable to roe products ranged from 36% to 60%, and they were slightly the major yolk protein vitellogenin or its derivatives (Schubring, lower than those of low-priced roe products (38% _ 72%). 2004). Fish egg denaturation temperatures of high-priced roe Cod eggs within the ovary are soft and can be activated to products (88.3℃ to 91.2℃) and those of low-priced products undergo a hardening process when fertilized or transferred to (88.5℃ to 92.0℃) were similar. High-priced roe products showed 346 C. Chen et al.

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