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*Corresponding author: Prof. Dr. Shanggui Deng, College of Food and Pharmacy, Zhejiang Ocean University, Zhoushan, Zhejiang 316000, China. Tel.: +86-580-2552539. Fax.: + 86-580-2552539. E-mail: dengshanggui@163.com Received: 23 August 2016; Revised: 13 March 2017; Accepted: 19 March 2017; Published Online: 29 March 2017 Yuan, et al.: Quality changes in squids chilled with Flake or Slurry Ice 340 Emir. J. Food Agric ● Vol 29 ● Issue 5 ● 2017 and thus reduces dehydration and oxidation events (V) the complete coverage of the seafood surface (Piñeiro et al., 2004; Aubourg et al., 2007; Kauffeld et al., 2010). Several studies suggest that slurry ice is a promising technology to improve the quality for a wide range of fish species (Piñeiro et al., 2004). For example, European Hake (Merluccius merluccius) (Losada et al., 2004), horse mackerel (Trachurus trachurus) (Losada et al., 2005), salmon (Oncorhynchus kisutch) (Rodríguez et al., 2008), seabass (Dicentrarchus labrax) (Cakli et al., 2006) and pink shrimp (Parapenaeus longirostris) (Huidobro et al., 2002). Although these advantages and applications of slurry ice are well known, few studies are available on the ability of slurry ice to extend the shelf-life of squid and enhance the quality parameters (Ommastrephes bartrami). Therefore, the objective of this study was to compare the effects of slurry ice and flake ice treatment on the microbiological, and chemical parameters of Squid (Ommastrephes bartrami). MATERIALS AND METHODS Preparation of flake ice (FI) and slurry ice (SI) An ice system (RF-1000-SP, JiangSu, China) was used to prepare the slurry ice (SI). The ice slurry mixture was consisted of 70% ice and 30% filtered seawater (salinity 3.0%).The temperature of the SI mixture ranged from -2.0 °C to -2.5°C. Flake ice (FI) was prepared using freshwater with a compact device (SM-F140AY65, HITACHI, Japan); the temperature of the FI ranged from 0 to 0.8°C. Fish material, processing, and sampling Fresh squid (Ommastephes bartrami) of approximately 10 to 15 cm body length and approximately 70.0 g body weight were provided by Zhejiang XingYe Company (Zhoushan, China). Squids were transferred to the laboratory within 30 min into aseptic bags and stored in zero degrees using portable refrigeration box. Upon arrival in the laboratory they were divided into two groups and placed in SI or FI at a ratio of 13 (fish to ice), before being stored in a refrigerated room at 0°C for 15 days. The FI and SI were renewed every day during the storage period. To determine the cooling curves for squid stored in SI and FI the temperature of different parts of squid was measured by multiplex temperature tester (JK-24U, JiangSu, China) every 0.5 min for 30min. For each chilling treatment, three carcasses without skin and cartilage were studied separately throughout the whole experimental period. Every three days, the samples were subjected to chemical and microbial analyses. Determination of salinity and moisture content To determine the salinity level in the squid muscles, about 10.0 g minced squid were mixed with 100 ml water (70 °C) and boiled for 15 min with shaking. The samples were homogenized for 1.0 min (high speed) before cooled to room temperature. Then the salinity content was measured using salinometer (YSI EC300, America) as described by Chinese National Standard GBT 12457-2008. Moisture content was determined by Automatic moisture meter (HG63, METTLER, Switzerland) according to Chinese National Standard (GB 5009-2010). 5.0 g minced squid were placed on the specimen disc with zero clearing, the moisture contents were measured directly and recorded, each sample was measured five times. Determination of total volatile basic nitrogen (TVB-N) Total Volatile Bases Nitrogen (TVB-N) was measured by automatic kjeldahl apparatus (FOSS-8400, Sweden). The results were expressed as mg N (100g)-1muscle. Squid samples prepared as described by (Aubourg et al., 1997) with slight modification. briefly, 10 g of squid sample was homogenized with 90ml of 0.6M perchloric acid solution for 1 min using a laboratory homogenizer. The homogenate was centrifuged at 10000 rpm for 10 min (4°C) and the supernatant was filtered through Whatman No. 1 filter paper. Microbiological analyses The total bacterial load was determined using pour-plate method. Samples (25.0 g) were transferred to a sterile blender Jar containing 225 ml of sterile 1% peptone water (PW) and homogenized for 2 min using a stomacher. Serial dilutions of each homogenate were carried out with 0.1% PW. Appropriate dilutions (1 ml) were plated onto plate count agar (GB/T 4789.1-2010, China). The plates were incubated at 30 °C for 48 h to enumerate the total bacteria count. Myofibrillar protein analyses To isolate myofibrils from muscle, about 10.0g minced flesh was homogenized in 100 ml of 20 mM Tris-maleic acid solution (containing 0.05 mM KCL, pH7.0). The homogenate was centrifuged at 10000rpm for 10 min (-4°C), the supernatant discarded and the sediment, containing most of myofibrillar was resuspended in 100ml of 20 mM Tris-maleic acid solution (containing 0.6 mM KCL, pH7.0) and centrifuged again for 5 min at 9000 rpm (-4 °C). The resulting supernatant that contains total myofibril protein solution, was measured by biuret method (Gornall et al. 1949). Determination of Ca2+-ATPase activity ATPase activities were determined as described by (Ooizumi and Xiong, 2004). To prepare the reaction Yuan, et al.: Quality changes in squids chilled with Flake or Slurry Ice Emir. J. Food Agric ● Vol 29 ● Issue 5 ● 2017 341 mixture the following chemicals were added: 0.5 M Trismaleic acid-containing buffer (pH 7.0), 0.1 M CaCl2, H2O, 20 mM ATP solution and myofibrillar proteins. The reaction mixture was incubated for 5 min at 30 °C in a water bath, and terminated by adding 1.0 ml of chilled 15% (w/v) trichloracetic acid (TCA) solution. The blank group was mixed with 1.0 ml 15% TCA solution to denature proteins before the reaction. 1.0 ml sulfuric acid molybdate, 0.5 ml metal and 2.5 ml water were added to 1.0 ml reaction mixture followed by incubated for 45 min at room temperature and then measured at 640 nm, 0.5 mM KH2PO4 was used as a standard. the following formula has been used to calculate Ca2+-ATPase Ca2+-ATPase = (A-B)/(t×C) Where (A) refer to 1.0ml phosphoric acid generated in the reaction solution (μM) ; (B) is phosphoric acid generated in the Blank (μM); (T) is the reaction time (min) and (C) is The content of enzyme in 1.0 ml reaction solution. Determination of total sulphur (SH) The method described by (Zhang et al., 2015) has been used to measure total SH content, briefly, 1.0 ml myofibril protein was added to 9.0 ml 0.2 M Tris-HCL buffer containing 8.0 M carbamide, 2% (w/v) sodium dodecyl sulfate (SDS) and 10 mM EDTA, (pH6.8). Thereafter, 4.0 ml of this mixture was added into 0.4 ml of 0.1% (w/v) DTNB solution (containing 0.2 M Tris-HCl, pH8.0) and further subjected to incubation at 40°C for 25min in a water bath. Then the absorbance of the mixture was measured at 412 nm using a spectrophotometer (U2800, HITACHI, Japan). A blank was prepared by replacing the sample with 0.6 M KCl. Total SH content was calculated by the following formula

Comparative evaluation of the quality changes in Squid (Ommastrephes bartrami) during flake and slurry ice storage