Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate edible films

Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate edible films

Accepted Manuscript Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate...

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Accepted Manuscript Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate edible films Olaia Martínez, Jesús Salmerón, Leire Epelde, M.Soledad Vicente, Carmen de Vega PII:

S0956-7135(17)30473-5

DOI:

10.1016/j.foodcont.2017.10.003

Reference:

JFCO 5812

To appear in:

Food Control

Received Date: 11 July 2017 Revised Date:

2 October 2017

Accepted Date: 4 October 2017

Please cite this article as: Martínez O., Salmerón Jesú., Epelde L., Vicente M.S. & de Vega C., Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate edible films, Food Control (2017), doi: 10.1016/j.foodcont.2017.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding resveratrol and coating with chitosan and alginate edible films

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Olaia Martínez*, Jesús Salmerón, Leire Epelde, Mª Soledad Vicente, Carmen de

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Vega

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Department of Pharmacy and Food Science, Faculty of Pharmacy, University of the

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Basque Country UPV/EHU, Paseo de la Universidad, 7. 01006, Vitoria-Gasteiz, Araba,

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Basque Country (Spain). *e-mail: [email protected]. Tel.: (0034) 945013470.

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Fax: (0034) 945013014.

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ACCEPTED MANUSCRIPT 1 Quality enhancement of smoked sea bass (Dicentrarchus labrax) fillets by adding 2

resveratrol and coating with chitosan and alginate edible films

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5 1. Introduction

Sea bass is a highly valued fish that has already demonstrated good attitude towards

7 smoking (Fuentes, Fernández-Segovia, Serra & Barat, 2010). Thus, it could be a good

The European sea bass (Dicentrarchus labrax) is a fusiform fish from the Moronidae

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8 alternative to traditionally smoked species such as salmon, trout or cod.

10 Family (Linnaeus, 1758). It is a lean fish (about 1-2% fat) and source of high biological 11 value protein (21%). It is also a source of selenium (8 ppm), D vitamin (2.3 ppm), 12 eicosapentaenoic acid

(0.44%) and docosahexaenoic acid (0.58%) (European

13 Commission, 2012). European Union is the first worldwide producer with a share of

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14 80%, aquaculture being the major production method. Spain is the second most 15 important country in sea bass production in the EU, with 16.231 t in 2015 (APROMAR,

17 market.

Fish smoking prolongs shelf life and helps to achieve desirable organoleptic

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16 2016). Adding value to this fish could have a direct and significant impact in the

19 characteristics. Smoking by conventional method is a process of difficult control and it 20 is a significant source of polycyclic aromatic hydrocarbons (PAHs) which remain in the 21 smoked foodstuffs (Drabova, Pulkrabova, Kalachova, Tomaniova, Kocourek & 22 Hajslova, 2013). Nowadays smoke flavourings constitute the safer technological 23 alternative to traditional methods (Arvanitoyannis & Kotsanopoulos, 2012). Depending 24 on their composition, some smoke flavourings may help to improve quality or sensory 25 characteristics, while others contribute to a better hygienic quality (Martínez, Salmerón,

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ACCEPTED MANUSCRIPT 26 Guillén & Casas 2007a, 2007b). It is important to find an equilibrated application 27 protocol which brings together both aspects. 28

However, semi-preserved fish products, such as smoked fish fillets, often have

29 obvious deficiencies to preserve an acceptable level of quality (Kramarenko et al.,

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30 2016). Recent studies have shown the great potential of certain active natural molecules 31 to improve the shelf life of seafood: tocopherols, ascorbic acid, erythrobic acid, 32 rosemary

and sage extracts, etc. This could also be the case of 5-[(E)-2-(4-

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33 hydroxyphenyl)ethenyl]benzene-1,3-diol (IUPAC) or trans-resveratrol. Resveratrol is a

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34 phytoalexin synthesized by several plants in response to adverse conditions such as 35 environmental stress or pathogenic attack (Arora & Strange, 1991). Functional 36 properties of this molecule have been broadly studied (Marchal, Pifferi & Aujard, 37 2013), but there is little research on its role as possible food quality preservative. It has 38 a high antioxidant potential thus, it may help to retard rancidity flavour appearance in

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39 products as fish. Nevertheless, it has low resistance against light and it is rapidly 40 oxidized (López-Hernández, Paseiro-Losada, Sanches-Silva & Lage-Yusty, 2007). A 41 possible solution is to protect this molecule with films or edible coatings that would

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42 help to preserve its physicochemical properties along the shelf life of the foodstuff in

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43 which it is added. In addition, the low presence of this molecule in the basic diet would 44 also increase (Zamora-Ros et al., 2016). 45

An edible coating is a film which envelops food and which can be consumed

46 together with it. They help to maintain the quality of coated products, to keep the 47 microbiological stability and the mechanical integrity (Fernández-Pan, Carrión-Granda 48 & Maté, 2014). Alginate and chitosan biopolymers can form invisible edible coatings, 49 each one with different properties, which might make it possible to prolong shelf life of 50 seafood while keeping good quality. Some authors have described alginate to increase

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ACCEPTED MANUSCRIPT 51 water barrier, prevent microbiological contamination and maintain functional properties 52 (Song, Liu, Shen, You & Luo, 2011). Chitosan-based films have good mechanical 53 properties, selective gas permeability (CO2 y O2) and exert antibacterial and antifungal 54 activity (Fernández-Saiz, Sánchez, Soler, Lagaron & Ocio, 2013). There are no reports

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55 of experiences coating European sea bass with chitosan or alginate, neither combining 56 these edible films with resveratrol and liquid smoke flavourings. 57

The principal objectives of the present study are first, to find an equilibrated protocol

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58 to treat sea bass fillets with liquid smoke flavourings; second, evaluate the quality

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59 effects of superficial resveratrol addition in smoked European sea bass fillets and third, 60 examine how alginate and chitosan coatings influence in the quality of the product. 61 62 2. Materials and Methods

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63 2.1. Preparation and treatment of fish samples

23 sea bass (Dicentrarchus labrax) from aquaculture origin and standard size (600-

65 800 g category) were obtained (Grupo Tinamenor, Spain). They were cleaned from skin

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66 and bones and filleted under hygienic handling conditions. Fillets were divided in 2 big 67 groups of 25 and 20 fillets (156.12 ± 14.02 g) in order to perform the experimental work

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68 in two consecutive phases. The first one was used to test the possible impact of 69 resveratrol addition on the quality and shelf life of the smoked product. The second 70 group of fillets was used to study the effect of the edible coatings. 71 2.1.1. Fish preparation 72

For the first experimental phase, five fillets were kept as the untreated control group

73 (U). The rest of the samples were salted. Salting was performed by immersion of the 74 fillets in saturated brine for 12 h at 4ºC. Five brines fillets were kept as the salted

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ACCEPTED MANUSCRIPT 75 control group (B). The rest of the samples were smoked by immersion in liquid smoke 76 Scansmoke 9015 (Azelis) during 30 s. After drying, three batches, of five fillets each, 77 were separated. One was kept as the smoked control group (S) and two groups were 78 treated with resveratrol (Monteloeder, Spain), 2.5 g and 5 g per fillet, and were named

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79 SR1 and SR2, respectively.

In the second experimental phase, five fillets were salted in saturated brine, as

81 described before, and smoked by immersion in liquid smoke mixture (30 s) formed by

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82 pouring Scansmoke 9015 (Azelis) and AFS-10 SOL (Amcan Ingredients Europa,

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83 France) flavourings in 1:1 proportion. This batch was kept as the smoked control group 84 (S). At the same time, a suspension was prepared using the liquid smoke mixture 85 described and resveratrol (Monteloeder, Spain). 20 g/L of this substance were 86 suspended by continuous stirring. This mixture was used to treat the rest of the samples. 87 This quantity was chosen supposing a 100 dm2 of fillet direct contact area with the

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88 liquid smoke containing resveratrol. 15 fillets were treated by immersion in this 89 suspension. After drying five of these fillets were kept as the smoked and resveratrol90 added group (SR). The last ten fillets were processed: five covered with the alginate

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91 coating and the five left with the chitosan coating, both described in the following

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92 section, and they were named SRA and SRQ, respectively.

All the fillets were packed under vacuum and stored in the dark at refrigeration

94 temperature (4+0.5 °C). Physicochemical, sensorial and microbiological analyses were 95 performed weekly for each batch along five weeks. 96 2.1.2. Alginate coating 97

Alginate films were prepared as described by Song et al., (2011) with food-grade

98 sodium alginate (E-401, Apasa, Spain) in a 1.5% (w/v) aqueous solution. The solution 99 was stirred at controlled temperature (80°C) until the mixture became clear and then it

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ACCEPTED MANUSCRIPT 100 was mixed with glycerin [1:10]. This first alginate-glycerine solution was cooled to 101 room temperature. Fillets were immersed for 1 min before soaking them in a 2% 102 calcium chloride solution (w/v). 103 2.1.3. Chitosan coating

Chitosan coating solution was prepared as described by Jeon et al. (2002) mixing

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105 10 g chitosan from crab shells (Sigma-Aldrich) with 1000 mL distilled water containing 106 10 g glacial acetic acid. The mixture was stirred at 40 ºC for 1 h. Glycerol was added at

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107 a level of 1.0 mL/g of chitosan and stirred for 10 min. Each fish fillet was immersed for

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108 30 s and then it was allowed to stand for a 2 min period followed by a second 30 s 109 immersion. 110 2.2. Chemical analyses 111

Samples for trimethylamine analyses were taken from the tail. Thiobarbituric acid

112 reactive sampling was done from the belly. Finally microbiological, sensory and texture

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113 experiments were performed with samples from the dorsal part of the fillet. Sampling 114 was performed in triplicate.

115 2.2.1. Determination of thiobarbituric acid reactive substances (TBARS)

TBARS were determined using a modified method based on the protocol proposed

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117 by Tocher et al. (2002).

To 20-30 mg sample 1.5 mL of 20% TCA (w/v) with 0.05 mL of 1% BHT in

119 methanol were added. Then, 2.95 mL of a freshly prepared 50 nM thiobarbituric acid 120 (TBA) solution were added and the mixture was heated in a boiling water bath for 10 121 min. The reaction mixture was chilled and centrifuged (2000 xg). The absorbance of the 122 supernatant was measured at 532 nm using a UV/VISmini–1240 Shimadzu 123 spectrophotometer. For quantification, standard solutions were prepared using 1,1,3,3-

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ACCEPTED MANUSCRIPT 124 Tetraethoxypropane (TEP) (Sigma-Aldrich). Results are expressed as mg MDA eq./Kg 125 of fish flesh. 126 2.2.2. Determination of trimethylamine content (TMA) 127

TMA determination was carried following a modification of the AOAC 971.14

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128 method (1995) proposed by Gökoglu, Cengiz & Yerlikaya (2004).

25 g sample were blended with 75 mL of 5% trichloracetic acid (Panreac) using a

130 homogenizer and it was filtered. Bases other than trimethylamine (Sigma-Aldrich) were

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131 fixed with 5 mL formaldehyde (20%). A 4 mL aliquot was transferred into test tubes

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132 and 1 mL formaldehyde, 10 mL anhydrous toluene and 3 mL K2CO3 (1:1) (v/w) were 133 added. The tubes were shaken and kept for phase separation. 5 mL from the top toluene 134 layer were taken and pipetted to a test tube. 5 mL picric acid (0.02%, w/v) (Panreac) 135 were added. The mixture was shaken and transferred to a spectrophotometric cell. 136 Absorbance was measured at 410 nm using a spectrophotometer (Shimadzu

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137 UV/VISmini-1240). Series of TMA standards between 0 – 13.6 ppm were used. 138 2.3. Bacteriological analysis 139

10 g samples of sea bass fillets were serially diluted (1:10) in sterile 0.1% peptone

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140 water using a stomacher (Masticator, IUL Instruments) for 2 min at room temperature.

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141 Serial dilutions were prepared and 1 mL samples of the corresponding dilutions were 142 planted by the pour plate method (AOAC, 2002) and incubated as follows. 143

Aerobic and anaerobic mesophilic and psycrophilic counts were determined in plate

144 count agar (PCA; OXOID). Aerobic mesophilics at 32°C for 72h, anaerobic mesophilics 145 incubated in anaerobic conditions at 32 °C for 72h, and psychrophilics at 7 ºC for 7 146 days. 147

Microbiological data were transformed into logarithms of the number of colony-

148 forming units (CFU/g).

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ACCEPTED MANUSCRIPT 149 2.4. Texture analysis and sensory evaluation 150 2.4.1. Instrumental texture analysis 151

A texture profile analysis (TPA) was obtained by means of a double compression

152 assay using a TA.XT2i texture analyzer (Stable Micro Systems, UK). Test was done in

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153 triplicate along the dorsal muscle of the fish fillet pressing a distance equivalent to 30% 154 of the sample height at a speed of 2 mm/s. The following parameters were obtained 155 from the force-time curves: hardness (g), adhesiveness (g·s), gumminess (g).

For the sensory analysis a panel was formed with five qualified assessors who had

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156 2.4.2. Sensory evaluation

158 previous experience in organoleptic analysis of smoked fish. The first stage of the 159 sensory assay had, basically, the objective of determining whether it was feasible to add 160 resveratrol to the sea bass fillets, taking into account organoleptic characteristics. 161 During this initial approach U, B, S, SR1 and SR2 samples were evaluated for possible

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162 off-flavours coming from resveratrol addition. At the same time, assessors were trained 163 in sea bass evaluation using commercial smoked sea bass samples as quality references. 164 In the process, evaluation method and discriminative attributes were selected. Finally,

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165 five parameters were selected by consensus: appearance, deteriorate fish odor, texture

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166 when handling (adhesiveness), texture in the mouth or when handling, in case it had 167 unpleasant odor or high microbiological counts found (firmness) and flavours in mouth 168 (fresh fish). This attributes were used to assess initial and final sensory characteristics of 169 samples S, SR, SRA and SRQ. 170

Samples corresponding to different treatments were finely sliced making cuts

171 diagonal to the surface of the fillet. They were presented to the panelists randomly 172 coded with a 3 digit numbers. Assessors were asked to score each attribute using a

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ACCEPTED MANUSCRIPT 173 structured five-point intensity scale. 2.5 were taken as rejection limit. Finally, they were 174 asked to give a global description for each sample. 175 2.5. Statistical analysis 176

Analyses were done in triplicate. Results are expressed as mean values ± standard

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177 deviation. IBM SPSS Statistics for Windows 22.0 was used for data analysis.

Differences in several physicochemical parameters between batches and along time

179 are characterized by ANOVA. Pearson correlation coefficient was calculated to study

TMA increase is commonly linked to microbial growth rate and, thus, it usually has

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180 relation between trimethylamine and microbiological growth.

182 an exponential growth interval preceded by an initial low rate and followed by a 183 maximum in which TMA concentration does not increase significantly any longer 184 (Heising, Van Boekel & Dekker, 2014). TMA development along storage time was 185 adjusted to an adapted sigmoid four parameter model in all the batches (Martínez et al.,

=

+(

). Parameters should be interpreted as follows: β0

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186 2012):



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187 is the initial concentration at time t = 0 (mg TMA-N per 100 g fish); βmax is the upper

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188 asymptote concentration (mg TMA-N per 100 g fish); β1 corresponds to the slope of the 189 linear increase; λ is the delay in time before the linear increase in concentration starts

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190 and t, time (days).

192 3. Results and discussion 193 3.1. First experimental phase: surface resveratrol addition 194 3.1.1. Chemical analyses 195

In general, untreated samples (U) showed the highest TBARS values. Salting and

196 smoking seemed to reduce TBARS significantly (p≤0.05) comparing to control group 197 (U). Resveratrol addition did also have a significant effect in TBARS reduction, which

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Salting is known to promote oxidation via iron displacement from haemoglobin

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203 molecule. Nevertheless, dry-salting has a greater effect comparing to brine salting, in 204 which the loss of heme proteins to the salting medium might cause a lowered lipid

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205 oxidation (Chaijan, 2011). Comparing TBARS values for B and S, they were higher in

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206 the first and second time points for the salted samples and otherwise from the third 207 measurement on. Overall differences between both batches could be due to samples 208 heterogeneity. On the other hand, maximum TBARS values in smoked samples 209 appeared one week later than in salted fillets. Liquid smoke addition might help to delay 210 secondary oxidation products as the liquid smoke used is rich in phenol compounds as

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211 quantified by Guillén and Ibargoitia (1996) and Guillén and Manzanos (1996). Finally, 212 superficial addition of resveratrol resulted in an efficient aid against oxidation, surely 213 caused by its high antioxidant potential, which was enhanced with greater addition in

Initial TMA levels were low for all the groups and were in a range from

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214 the SR2 samples.

216 0.443±0.015 mg TMA-N/100 g to 0.678±0.015 TMA-N/100 g (Table 1). U samples 217 showed the highest TMA values (p≤0.05) along five weeks storage period. Maximum 218 trimethylamine levels were registered by the third week of analysis in all the groups. In 219 general, values for brined fillets were significantly lower (p≤0.05) than for smoked ones 220 (S). SR2 batch had the overall lowest values for TMA (p≤0.05). Table 2 shows β0, βmax, 221 β1 and λ parameter values for the model proposed, in which estimated TMA values were 222 highly correlated with observed concentrations (R2: 0.926-0.986).

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Initial values for TMA were similar to those previously reported for refrigerated sea

224 bass in anaerobic conditions (Masniyom, Benjakul & Visessanguan, 2002). However, 225 our TMA maximum values remained lower all along the storage period. U fillets were 226 the only ones showing a maximum above 5 mg TMA-N/100g, taken as limit for

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227 rejection. A positive correlation (p≤0.05) was found between TMA and microbial 228 counts. TMA is produced by microbial reduction from trimethylamine oxide (TMAO) 229 and it is usually interpreted as a hygienic indicator. Moreover, TMAO can also be

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230 broken down by enzymatic autolysis, forming dimethylamine (DMA) and formaldehyde

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231 (FA), especially in gadiform species during frozen storage (Seibel & Walsh, 2002). 232 Raeisi, Sharifi-Rad, Quek, Shabanpour and Sharifi-Rad, (2016) described how TMA 233 content in rainbow trout was lower in the presence of fruit and seed extracts exhibiting 234 antimicrobial and antioxidant properties. Badii and Howell (2002) demonstrated that in 235 the presence of antioxidants FA formation was enhanced during frozen storage of cod.

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236 This would reduce microbial TMA formation and it would explain lower TMA values 237 in SR2 fillet group (p≤0.05).

238 3.1.2. Bacteriological analysis

U samples gave higher counts all through the experiment than any other batch for

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240 total viable count (TVC), psychrophilic and especially, for anaerobic microorganisms 241 (Fig. 1). The rest of the groups had similar counts, which suggests that the reduction in 242 microbial counts from U to the other batches may be due to salting and smoking 243 processes. Masniyom, Benjakul and Visessanguan (2002) had TVC around 106 CFU/g 244 after 18 days of refrigerated storage of sea bass slices under anoxic and refrigerated 245 conditions, while in our experiment this level was achieved in about ten days in U 246 fillets. Nevertheless, other authors have recorded higher counts than us regarding

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Finally, some authors have shown the potential of trans-resveratrol to damage cell

250 membrane integrity of lactic acid bacteria (Mora-Pale et al., 2015). However, results do

252 3.1.3. Texture analysis and sensory evaluation 253

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251 not suggest any significant effect of resveratrol on microbial counts.

Hardness had a decaying tendency in all the batches (Fig. 2). SR2 was the group that

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254 presented the highest hardness (1628.415±192.353 gram-force, g) from the beginning of

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255 the storage time and all along the first two weeks. It was followed by SR1 during the 256 first week, after which the hardness of this batch decayed below the values for brined 257 samples. B group went from 1281.360±154.360 g (initial values) to 862.350±219.92 g 258 (values register in the last week). U and S samples showed the lowest hardness all along 259 the assay. For adhesiveness, initial values ranged from 15.88±3.453 g·s to 35.683±2.232

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260 g·s. U, B and S samples showed a clear increasing tendency along storage time for this 261 parameter; values for these three batches were between 55.64±5.498 g·s and 262 68.816±12.378 g·s in the last analysis, higher (p≤0.05) than adhesiveness for SR1 and

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263 SR2. Regarding gumminess, U samples showed the lowest values comparing with S and

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264 B samples. Brined samples had the highest gumminess values from the three groups 265 (p≤0.05). SR1 and SR2 presented initial similar gumminess values, the highest among all 266 the groups, but started to descent from the day 10 and 18 on, respectively. 267

Treatment with Scansmoke gave softer textures than only brined samples. This effect

268 was previously described and linked to the phenol content of the liquid smoke and its 269 capacity to hold water molecules (Martínez et al., 2007a; 2007b). 270

Szczesniak (2002) defined gumminess as the energy required to disintegrate a semi-

271 solid food to a state ready for swallowing that would be correspondent to the denseness

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ACCEPTED MANUSCRIPT 272 that persists throughout mastication. It is a term applicable to semi-solid food which has 273 been previously applied to instrumental texture evaluation of fish flesh considering it 274 accomplishes this criterion (Martínez et al., 2007a; Chéret, Chapleau, Delbarre-Ladrat, 275 Verrez-Bagnis & de Lamballerie, 2005). Chéret et al. (2005) described significant decay

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276 in this parameter along 14 days cold storage of sea bass fillets. Similar tendency was 277 observed in our results. Finally, hardness and gumminess values were lower but this can 278 be due to differences in raw material characteristic and because our first measurements

It is noteworthy that superficial resveratrol addition in the quantities assayed was

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279 were made two days after processing.

281 easily appreciated in the fillet surface and gave the samples a disgusting aspect. 282 Nevertheless, assessors did not find any other distinctive attribute coming from 283 resveratrol addition to sea bass fillets, either in SR1 or SR2 batch (2.5 g and 5 g per 284 fillet, respectively).

Some author as Gaudette and Pickering (2011) reported that fortifying wine with

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286 resveratrol (200 µg/mL) did not alter its aroma and flavor, but it induced bitterness in 287 Riesling wines. In Cabernet Sauvignon wines only color intensity appeared to be

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288 altered. Koga, Lee and Lee (2016) did not find significant differences in consumers

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289 liking after addition of resveratrol in bars and gummies. 290 3.2. Second experimental phase: resveratrol added as suspended molecule in smoke 291 flavouring and protected with chitosan or alginate edible coating 292 3.2.1. Chemical analyses 293

Control batch, S, gave the highest TBARS values, after 7 days of refrigerated storage

294 (2.371+0.068 mg MDA/Kg). The rest of the samples had their maximum values in the 295 third week; the lowest maximum (1.273+0.016 mg MDA/Kg) was obtained for SRQ. In

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Antioxidant addition in the liquid smoke mixture (20 g/L) was enough to cause a

299 delay in the apparition of maximum values in oxidation secondary products. This was a

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300 lower dose comparing to quantities employed to study potential health benefits of 301 resveratrol (Marchal et al., 2013), but theoretically similar to the dose at which 302 resveratrol has proved to have an antioxidant potential and scavenging activity (Gülçin,

In general, TMA values increased significantly (p≤0.05) with storage time (Table 1).

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303 2010).

305 Resveratrol suspended in liquid smoke had no effect on TMA comparing to S fillets. 306 This batch (just smoked) presented lower TMA values comparing with smoked samples 307 from the trial in which resveratrol was superficially added. Lowest maximum data 308 (p≤0.05) were described in the group coated with chitosan (SRQ) and highest for the

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309 samples treated with alginate (SRA).

As when resveratrol was superficially added, TMA concentration followed a non

311 linear tendency with time (Table 2). Resveratrol suspension in the liquid smoke did not

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312 have any apparent effect in the parameters estimated for the model; both S and SR

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313 fillets showed similar results, usually with intermediate values between SRA and SRQ. 314 Slope of the linear portion of the curves (β1) was the lowest for SRQ batch and the 315 greatest for SRA, indicating faster TMA increase for this group. At the same time, the 316 fillets coated with alginate had the highest βmax values and had the shortest delay time 317 (λ). The composition of the smoke and the chitosan coating seem to contribute to a 318 better quality of the sea bass fillets in this second experiment, attending to results 319 obtained in the analysis of TMA. 320 3.2.2. Bacteriological analysis

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Pearson correlation analysis between trimethylamine and microbiological viable

322 counts. Correlations were significant at α= 0.001 level for most of the batches with the 323 exception of SRQ, which only showed this tendency for psychrophilic microorganisms. 324 This could suggest that TMA origin in chitosan coated fillets is preferentially autolytic.

Unlike in the previous experiments, acceptable limits of 106 CFU/g (ICMSF, 1986)

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326 were not reported until the last analysis (Fig. 3). Mesophilic, anaerobic and 327 psychrophilic bacteria counts showed similar pattern along storage time. Significant

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328 (p≤0.05) increasing values were described for S, SR and SRA with the highest counts at

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329 the fourth week for which most of the values were between 105 and 106 CFU/g. This 330 supposes an important improvement in shelf life respect the first assay in which critical 331 counts were surpassed 14 days before. For mesophilic and anaerobic bacteria highest 332 counts were obtained for the alginate coated samples, 5.8+0,02 log CFU/g and 333 6.71+0,23 log CFU/g, respectively. Chitosan clearly protected fillets against bacterial

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334 growth. Counts were under 101 CFU/g at most of the analysis time point during 335 refrigerated vacuum storage. Counts were punctually higher at time 3 for mesophilic 336 and at time 3 for anaerobic bacteria, this may be due to random contamination.

Addition of AFS-10 SOL (high carbonyl content) to the liquid smoke may have

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338 contributed to a lower microbiological growth, as described in previous articles 339 (Martínez et al., 2007a; 2007b), and thus, to lower TMA amounts. Alginate coating 340 does not exhibit antibacterial properties by itself but has been described to delay 341 microbial growth when applied in fish stored in aerobic conditions, because it acts as a 342 barrier against oxigen transfer (Song et al., 2011). In vacuum conditions, this effect 343 would be negligible. Finally, chitosan has a known antimicrobial potential. Günlü and 344 Koyun (2013) had similar results, reporting a 20 days longer shelf life for chitosan

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ACCEPTED MANUSCRIPT 345 wrapped sea bass fillets vacuum-packaged and stored at 4 ºC compared to control 346 samples, without chitosan, for which shelf life was estimated in 5 days. 347 3.2.3. Texture analysis and sensory evaluation 348

Main texture results are shown in Fig. 4. Regarding hardness, S samples were the

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349 softest ones during the previous three analyses (2 weeks). SR samples, on the contrary, 350 maintained high values of hardness during the first two analyses, significantly not 351 different from SRQ group, and started to decline by day 18.

These results would suggest, similarly to the first experiment, that resveratrol

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353 addition has a hardening effect on fish flesh. Pastor, Sánchez-González, Chiralt, Cháfer 354 and González-Martínez (2013) have described increase in rigidity in a methylcellulose 355 composite film after resveratrol addition and propose that this could happen due to the 356 formation of crystalline structures. However, this seemed to be dependent of the matrix 357 in which it is added. Polyphenols are known to react under oxidizing conditions with

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358 side chain amino groups of peptides, leading to formation of cross-links in proteins 359 giving a structure of grater mechanical strength (Strauss & Gibson, 2004). Several 360 authors have reported similar results implying interactions between myofibrillar fish

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361 proteins and phenolic compounds (Prodpran, Benjakul & Phatcharat, 2012; Balange &

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362 Benjakul, 2009).

In general, initial organoleptic quality was good for all the samples. Aroma and

364 flavour coming from mixing both liquid smokes was highly appreciated by assessors. 365 The method based on suspending resveratrol in the liquid smoke mixture succeeded to 366 mask its presence. Acceptability of all the samples decayed in the last analysis, mainly 367 related to appearance evaluation. Superficial moisture due to liquid loss of the fillets 368 became very evident at this stage and this was pointed out and negatively evaluated by 369 assessors. Significant differences between the batches became more evident during last

15

ACCEPTED MANUSCRIPT 370 week. Lower adhesiveness was detected in both coated batches, which were also judged 371 as firmer than S and SR. Deterioration flavour notes were detected by the panellists for 372 the SRA group in higher proportion than the rest of the batches. Subsequent lower score 373 in “Fresh fish flavour” attribute was recorded for the alginate coated samples. Samples

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374 without coatings have more intense smoke odour which might have masked 375 deterioration notes. 376

In this study European sea bass probed to be an alternative raw material for new

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377 3. Conclusions

379 slightly-preserved smoked fish fillets products. The mixture of two liquid smokes with 380 different composition exhibited the antibacterial properties of one of them without any 381 sensory quality loss. Salting and liquid-smoking lead to an important improvement in 382 hygienic quality, when the adequate mixture is chosen. Suspending resveratrol in the

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383 liquid smoke in which fish fillets are immersed for treatment was effective, as its effects 384 were maintained while its presence was not as obvious as when it is superficially added. 385 Resveratrol did not alter smoked sea bass sensory characteristics and it demonstrated to

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386 be a good protector against chemical deterioration of sea bass fillets. Results suggested

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387 that it also plays a role in texture development of fish fillets, in which interactions 388 phenol-proteins might be involved. This aspect should be studied further. Alginate 389 protected fillets against oxidation but did not inhibit bacteria growth and was linked to 390 “fish deterioration favours” during the last sensory evaluation. It was chitosan which 391 produced the greatest reduction in viable counts; almost a complete inhibition of the 392 growth of mesophilic, psychrofilic and anaerobic bacteria. 393 394 Acknowledgements

16

ACCEPTED MANUSCRIPT 395 This work was supported by the Basque Government [grant number SA-2011/00117; 396 competitive call for research projects (SAIOTEK)]. 397 398 References

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intake in Europe: the European Prospective Investigation into Cancer and Nutrition

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(EPIC) study. European Journal of Nutrition, 55(4), 1359-1375.

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Table 1.

2

Mean values and standard deviations for the TMA and TBARS measured during the first experimental phase for batches U, B, S, SR1, SR2 (A)

3

and measures during the second experimental phase for groups S, SR, SRA, SRQ (B). Different letters (a, b, c, d) indicate significant differences

4

(p≤0.05) between batches in each time point. Different numbers (1, 2, 3, 4, 5) indicate significant differences (p≤0.05) between time points for

5

each batch.

6

A)

Storage Time (weeks)

U

B

0

0.620 ± 0.039 b,c;4

TBARS

1

(mg Eq. MDA/kg fish flesh)

SR1

SR2

0.167 ± 0.011 d;3

0.601 ± 0.038 c;4

0.723 ± 0.046 a;4

0.675 ± 0.043 a,b;1

4.335 ± 0.276 b;3

4.969 ± 0.316 a;1

3.011 ± 0.191 c;2

2.169 ± 0.138 d;1

0.482 ± 0.031 e;2

2

6.576 ± 0.418 a;1

2.608 ± 0.166 b;2

6.122 ± 0.389 a;1

1.815 ± 0,115 c;2,3

0.372 ± 0.024 d;3

3

5.372 ± 0.342 a;2

2.403 ± 0.153 c;2

3.123 ± 0.199 b;2

1.941 ± 0.123 d;2

0.288 ± 0.018 e;3

4

6.177 ± 0.393 a;1

2.318 ± 0.147 b;2

2.400 ± 0.153 b;3

1.653 ± 0.105 c;3

0.298 ± 0.019 d;3

0

0.678 ± 0.015 a;5

0.449 ± 0.020 c;5

0.443 ± 0.015 c;5

0.589 ± 0.018 b;4

0.590 ± 0.022 b;4

TMA

1

2,366 ± 0.015 a;4

2.159 ± 0.015 b;2

2.127 ± 0.013 c;4

2.037 ± 0.016 d;3

2.037 ± 0.015 d;2

(mg TMA-N/100g fish flesh)

2

5.577 ± 0.016 a;1

2.910 ± 0.076 d;1

3.295 ± 0.085 c;2

3.709 ± 0.094 b;1

2.696 ± 0.093 e;1

3

4,210 ± 0.104 a;3

1.463 ± 0.046 d;4

2.748 ± 0.073 c;3

3.185 ± 0.082 b;2

1.501 ± 0.046 d;3

4

4.747 ± 0.116 a;2

1.974 ± 0.056 d;3

3.664 ± 0.093 b;1

3.279 ± 0.084 c;2

1.539 ± 0.047 e;3

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Parameters

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B) S

SR

SRA

SRQ

0

1.293 ± 0.035 a;2

0.770±0.032 c;2

0.947±0.013 b;2

0.623±0.032 d;2

TBARS

1

2.371 ± 0.068 a;1

0.795±0.071 b;2

0.490±0.008 d;4

0.545±0.011 c;2

(mg Eq. MDA/kg fish flesh)

2

0.522 ± 0.008 c;4

0.786±0.012 a;2

0.631±0.028 b;3

0.503±0.009 c;3

3

0.760 ± 0.008 d;3

1.576±0.025 b;1

1.667±0.044 a;1

1.274±0.016 c;1

4

0.332 ± 0.010 b;5

0.628±0.007 a;3

0.288±0.015 c;5

0.216±0.052 d;4

0

0.578±0.023 b;3

0.437±0.056 c;4

0.851±0.033 a;4

0.400±0.081 d;4

TMA

1

0.574±0.033 c;3

0.817±0.030 b;3

1.181±0.045 a;3

0.746±0.076 b;3

(mg TMA-N/100g fish flesh)

2

1.923±0.006 b;2

1.505±0.059 c;2

3.207±0.121 a;2

1.000±0.043 d;2

3

2.260±0.132 c;1

2.689±0.085 b;1

4.355±0.132 a;1

1.796±0.068 d;1

4

2.169±0.155 c;1

2.636±0.130 b;1

4.297±0.005 a;1

1.889±0.057 d;1

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(A) U, untreated; B, brines; S, brined and smoked; SR1, brined, smoked and resveratrol added superficially in a proportion of 2.5 gr./fillet; SR2, brined, smoked and resveratrol added superficially in a proportion of 5 gr./fillet. (B) S, brined and smoked fillets; SR, brined and treated with a liquid smoked suspension containing resveratrol (20 gr/L); SRA, treated as SR and coated with alginate; SRQ, treated as SR and coated with chitosan.

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Storage Time (weeks)

Parameters

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Table 2.

2

Parameters for the model

3

development along five weeks in the samples analyzed on the (A) first experimental

4

phase and (B) second experimental phase.

=

+(



∗ )

which explains TMA

)

A) λ -29.360 0.563 1.870 -30.643 1.415

β1 2.900 0.258 0.119 3.071 0.193

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βmax 4.511 2.590 3.468 3.392 2.598

Samples β0 λ β1 S -1.869 -4.527 0.318 SR -2.106 -4.161 0.231 SRA -2.818 -5.987 0.460 SRQ -1.569 -2.054 0.123 Numbers in bold are significant (p≤0.05)

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R2 0.986 0.946 0.926 0.943 0.978

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β0 -3.834 -8.435 -27.961 -2.802 -14.18

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Samples U B S SR1 SR2

βmax 2.261 2.553 3.546 1.847

R2 0.962 0.817 0.741 0.804

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Fig. 1. Changes in total viable counts (CFU/g) for (A) mesophilic, (B) anaerobic and (C) psychrotophic microorganisms during cold storage (4ºC) of vacuum packed sea bass fillets: U, control samples; B, brined samples; S, liquid smoked samples; SR1, fillets

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resveratrol addition (5 g/fillet).

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Fig. 2. Changes in A) hardness (g), B) adhesiveness (g·s) and C) gumminess registered for sea bass fillets along five weeks of vacuum refrigerated (4ºC) storage. Samples

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differences (p≤0.05) between time points for each batch (for adhesiveness some

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Fig. 3. Changes in total viable counts (CFU/g) for (A) mesophilic, (B) anaerobic and (C) psychrotophic microorganisms during cold storage (4ºC) of vacuum packed sea bass

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Fig. 4. Changes in A) hardness (g) and B) adhesiveness (g·s) registered for sea bass fillets along five weeks of vacuum refrigerated (4ºC) storage. Samples were: S, smoked

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fillets; SR, fillets treated with resveratrol suspended in liquid smoke mixture; SRA, fillets treated like SR and coated with alginate and, SRQ, fillets treated like SR and coated with chitosan. Different letters (a, b, c) indicate significant differences (p≤0.05) between batches at the same time point and different numbers (1, 2, 3) indicate significant differences (p≤0.05) between time points for each batch.

ACCEPTED MANUSCRIPT 1

Highlights Sea bass is a good raw material for slightly-preserved smoked fish products

3

Resveratrol did not alter sensory quality of smoked sea bass

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Resveratrol antioxidant effect was enhanced when coated with alginate or chitosan

5

Chitosan coating improved greatly the hygiene of liquid smoked sea bass fillets

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