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Enzymatic Activity During Frozen Storage of Atlantic Horse Mackerel (Trachurus Trachurus ) Pre-Treated by High-Pressure Processing

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Enzymatic Activity During Frozen Storage of Atlantic Horse Mackerel (Trachurus Trachurus ) Pre-Treated by High-Pressure Processing Enzymatic Activity During Frozen Storage of Atlantic Horse Mackerel (Trachurus trachurus ) Pre-treated by High-Pressure Processing Fidalgo, L. G., Saraiva, J. A., Aubourg, S. P., Vázquez, M., & Torres, J. A. (2015). Enzymatic Activity During Frozen Storage of Atlantic Horse Mackerel (Trachurus trachurus) Pre-treated by High-Pressure Processing. Food and Bioprocess Technology, 8(3), 493-502. doi:10.1007/s11947-014-1420-9 10.1007/s11947-014-1420-9 Springer Accepted Manuscript http://cdss.library.oregonstate.edu/sa-termsofuse 1 Enzymatic activity during frozen storage of Atlantic horse 2 mackerel (Trachurus trachurus) pre-treated by high pressure 3 processing 4 5 6 7 Liliana G. Fidalgo1, Jorge A. Saraiva1*, Santiago P. Aubourg2, Manuel Vázquez3, J. 8 Antonio Torres4 9 10 11 12 1Research Unit of Organic Chemistry, Natural and Agro-food Products (QOPNA), 13 Chemistry Department, Aveiro University, Campus Universitário de Santiago, 3810- 14 193 Aveiro, Portugal. 15 2Department of Food Technology, Instituto de Investigaciones Marinas (CSIC), 36208- 16 Vigo, Spain 17 3Department of Analytical Chemistry, Faculty of Veterinary Science, University of 18 Santiago de Compostela, 27002-Lugo, Spain 19 4Food Processing Engineering Group, Department of Food Science & Technology, 20 Oregon State University, Corvallis, OR 97331, USA 21 22 23 * Corresponding author: Tel.: +351 234401513; fax: +351 234370084. 24 Email address: [email protected] (J. A. Saraiva) 1 25 Abstract 26 27 The assessment of enzymatic activity on Atlantic horse mackerel (Trachurus trachurus) 28 during frozen storage was carried out in samples pre-treated by high pressure processing 29 (HPP) combinations of 150, 300 and 450 MPa with 0, 2.5 and 5 min holding time 30 (untreated samples were used as controls). The activities of four enzymes (acid 31 phosphatase, cathepsins B and D, and lipase) in fish muscle were quantified during 32 accelerated storage conditions (up to 3 months at 10 ºC). The experimental data were 33 fitted to second order polynomial models to determine the effect of pressure level, 34 holding time, and frozen storage time on these enzymes activities, and to identify 35 conditions of maximum/minimal enzyme inactivation. Acid phosphatase and cathepsins 36 (B and D) activities were significantly (p < 0.05) influenced by HPP, showing 37 behaviours during frozen storage different from control samples. Acid phosphatase and 38 cathepsin B activities decreased (p < 0.05) with HPP treatments, being this effect more 39 intense for cathepsin B, particularly at 450 MPa. Regarding cathepsin D, the activity 40 increased (p < 0.05) at intermediate pressure (300 MPa) and decreased (p < 0.05) at 41 higher pressure (450 MPa). During frozen storage, cathepsin D enzymatic activity 42 tended to increase over time indicating activity recovery of these enzymes. Although a 43 predictive model for its activity was not acceptable, the increase in lipase activity during 44 storage was the most pronounced trend observed. 45 46 Keywords: High pressure processing, frozen storage, Trachurus trachurus, acid 47 phosphatase, cathepsins, lipase. 48 2 49 Introduction 50 51 Freezing and frozen storage are widely employed to retain the properties of fish 52 before consumption. However, quality is lost during frozen storage due to texture, 53 flavour and colour deterioration (Matsumoto 1979) caused by several factors including 54 the activity of endogenous enzymes. In fresh fish muscle, lysosomal enzymes, such as 55 cathepsins and acid phosphatase, play an important role in myofibrillar and connective 56 tissue degradation (Ladrat et al. 2003; Chéret et al. 2007; Hultmann et al. 2012). For 57 instance, Yamashita and Konagaya (1991) observed that cathepsin B hydrolyses 58 important myofibrillar proteins, including connetin, nebulin and myosin, causing a 59 drastic degradation of the muscle structure and consequently quality loss. These muscle 60 enzymes could be used as final quality indicators (Toldrá and Flores 2000). During 61 frozen storage, several hydrolytic enzymes are released and can cause fish muscle 62 quality loss. Burgaard and Jørgensen (2011) showed that frozen storage temperature did 63 not seem to affect rainbow trout (Oncorhynchus mykiss) cathepsin D activity. On the 64 other hand, Nilsson and Ekstrand (1995) observed that in the same fish species, frozen 65 storage temperature affected the integrity of lysosomal membranes, resulting in an 66 increase of lysosomal enzymes leakage and thus increased β-N-acetylglucosaminidase 67 activity when stored at 18 ºC instead of 40 ºC. 68 Lipolysis occurs extensively in fish muscle post-mortem and is associated with quality 69 deterioration in the frozen tissue (Shewfelt 1981). Lipase activity was reported to be the 70 principal cause for the formation of free fatty acids during albacore (Thunnus alalunga) 71 frozen storage (Gallardo et al. 1989) and was reported by Geromel and Montgomery 72 (1980) to be released from the lysosomes during frozen storage of trout (Salmo 73 gairdneri). Also, it has been shown that free fatty acids interact with proteins leading to 74 texture deterioration during frozen storage (Mackie 1993). 75 High pressure processing (HPP) has been shown to inactivate microorganism and 76 enzymes extending the shelf-life of many food products, while retaining high levels of 77 quality (Yordanov and Angelova 2010; Mújica-Paz et al. 2011). HPP can inactivate 78 enzymes by disrupting the bonds that determine their secondary, tertiary and quaternary 79 conformations, without affecting the covalent bonds in the primary structure. According 80 to Ashie and Simpson (1996), pressures up to 300 MPa decreased cathepsin C, 81 collagenase, chymotrypsin and trypsin-like enzymes activities in extracts from fresh 82 bluefish (Pomatomus saltatrix) and sheephead (Semicossyphus pulcher). Chéret et al. 3 83 (2005b) observed that pressures up to 500 MPa increased the cathepsins B, H, and L 84 activities in fresh sea bass (Dicentrarchus labrax L.) fillets. However, Teixeira et al. 85 (2013) obtained different results for acid phosphatase, cathepsin D and calpain from the 86 same fresh fish species (sea bass), having observed that activity reduction was maximal 87 at 400 MPa. 88 Atlantic horse mackerel (Trachurus trachurus) is a medium-fat species abundant in 89 the Atlantic Northeast that has recently drawn a great deal of commercial interest 90 (Aubourg et al. 2004). In recent previous work, valuable information was obtained 91 concerning HPP application in this fish species to inhibit lipid damage development 92 during subsequent frozen storage. HPP pre-treatments (150-450 MPa for 0-5 min) led to 93 a marked inhibition of lipid hydrolysis on frozen Atlantic horse mackerel (Torres et al. 94 2013). In addition, while slight differences were observed on water holding capacity, 95 colour, and texture, sensorial analysis after cooking revealed no identifiable significant 96 differences (Torres et al. 2014). Concerning the activity of endogenous enzymes, only 97 few reports on the effect of HPP pre-treatments followed by refrigeration storage have 98 been published, while published work on the effect of HPP pre-treatments during 99 subsequent frozen fish storage is practically non-existent. The effect of these pre- 100 treatments on enzymatic activities during the frozen storage of another species, Atlantic 101 mackerel (Scomber scombrus), was recently published by Fidalgo et al. (2014a). Thus, 102 the aim of this study was to investigate the effect of HPP pre-treatments on the activity 103 of acid phosphatase, cathepsins (B and D) and lipase in frozen Atlantic horse mackerel 104 during 3 months of frozen storage under accelerated storage conditions (–10 ºC). 105 106 107 Materials and Methods 108 109 Chemicals 110 111 Sodium dodecyl sulfate (SDS), trichloroacetic acid (TCA), acetic acid, trizma 112 hydrochloride (Tris-HCl), dithiothreitol (DTT), 2-bis-(2-hydroxyethyl)-amino-2- 113 (hydroxymethyl)-1,3-propanediol (Bis-Tris), ethylenediaminetetraacetic acid (EDTA), 114 p-nitrophenol, thymolphtalein, sodium hydroxide (NaOH), citric acid, trisodium citrate 115 and L-tyrosine were obtained from Sigma-Aldrich (Steinheim, Germany). Other 116 chemicals, such as potassium hydroxide (KOH) and sodium acetate were acquired from 4 117 Panreac Quimica S.L.U. (Barcelona, Spain). Substrates used in the determination of 118 enzymatic activity, e.g., p-nitrophenyl phosphate disodium salt hexahydrate (p-NPP, 119 #N22002), Z-arginine-arginine-7-amido-4-methylcoumarin hydrochloride (Z-Arg-Arg- 120 7-AMC HCl, #C5429), hemoglobin from bovine blood (#H2625), and olive oil 121 (#O1514) were also purchased from Sigma-Aldrich. 122 123 124 Raw material, processing and storage conditions 125 126 Fresh Atlantic horse mackerel (Trachurus trachurus), caught near the Bask coast in 127 Northern Spain (Ondarroa Harbour, Bizkaia, Spain), were transported under 128 refrigeration to the AZTI Tecnalia (Derio, Spain) pilot plant for HPP treatment. Whole 129 fish individuals (25-30 cm and 200-250 g range) were placed in flexible polyethylene 130 bags (three individuals per bag) and vacuum sealed at 400 mbar. 131 Whole fish were treated by HPP in a 55-l high pressure unit (WAVE 6000/55HT; 132 NC Hyperbaric, Burgos, Spain) at 150, 300 and 450 MPa for 0, 2.5 and 5 min holding 133 times following the experimental design presented in Table 1. Experiments with 0 min 134 pressure holding time were carried out to study the effect of the pressure come-up and 135 depressurising time. Non-pressure treated samples (T0, untreated control) were also 136 studied. The pressurising medium was water applied at 3 MPa/s, yielding come up times 137 of 50, 100 and 150 s for treatments at 150, 300 and 450 MPa, respectively; while 138 decompression time took less than 3 s. Pressurising water was cooled down to maintain 139 room temperature (20 ºC) conditions during HPP treatment. HPP-treated and control 140 samples were frozen in still air at 20 ºC for 48 h before storage at 10 ºC for sampling 141 after 0, 1 and 3 months. A higher temperature (10 ºC) than the one employed 142 commercially for frozen storage (18 ºC) was chosen to accelerate time effects and thus 143 reduce the duration of experiments.
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