A the Effects of Essential Oils Addition

8/13/2019 A the Effects of Essential Oils Addition

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The effects of essential oils addition on the quality of wild and farmed sea bream ( Sparus1

aurata ) stored in ice 2

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Mourad Attouchi 1 and Saloua Sadok 1*4

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Affiliation:1Institut National des Sciences et Technologies de la Mer7

Centre La Goulette, La Goulette, 2060, Tunisia.8

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*Corresponding author: Tel/fax: +2167173584813

Email address: [email protected] (S. Sadok)14

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Manuscript


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Abstract28

The preservative effect of laurel ( Laurus nobilis ) and cumin (Cuminum cyminum ) essential29

oils (EOs) on fresh vacuum packed (VP) wild and farmed sea bream (Sparus aurata ) fillets30

was evaluated during ice storage by microbiological, physicochemical and electrophoretic31

analyses. In the present study, wild (W) and farmed (F) fillets treatment included the32

following lots: control vacuum packaged samples (WV and FV), VP with added EOs (0.5%33

v/w) of cumin (WVC and FVC), and of laurel (WVL and FVL). Wild and cultured fish were34

found to differ significantly in their muscle proximate compositions irrespective of fillet35

treatments with particularly higher fat and carbohydrates contents in farmed sea bream (4.8236

and 0.32g/100g respectively; vs. 1.53 and 0.22g/100g in wild fish). The treatment of wild and37

farmed sea bream fillets with laurel or with cumin EOs induced a decrease in bacterial growth38

by ca. 0.5 to 1 log cfu/g and in lipid oxidation by ca. 40% of TBA value; extending the shelf39

life of fish fillets by approximately 5 days of ice storage. However, the addition of EOs to VP40

fillets resulted in a reduced liquid holding capacity (LHC) throughout ice storage suggesting41

an early proteolysis initiation confirmed by the myofibrillar and sarcoplasmic electrophoretic42

profiles. Laurel and cumin EOs as natural and efficient antibacterial and antioxidant43

compounds can be used in conjunction with VP to enhance ice stored sea bream quality.44

Keywords: Sea bream; essential oil; laurel; cumin; vacuum-packaging; quality; liquid45

holding; protein electrophoresis 46

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Introduction53

Wild and farmed fish are gaining an increased importance as healthy food, because54

numerous species have been identified as rich in therapeutically important polyunsaturated55

fatty acids, easily digestible proteins, in addition to vitamins and various other nutrients.56

Concurrently, important changes in pattern of food consumption are occurring including57

preference for safe and minimally processed foodstuffs. In the case of fish and seafood, food58

market analysts have estimated that changes will concern mainly changes in commodities; the59

overall tendency of seafood supply will show over the next decades, an increase of filleted60

and prepared/preserved fish and shellfish (Failler, 2007). Changes in food consumption also61

included the preference of lightly preserved food products with natural additives (Burt, 2004).62

For instance, spices and herbs extracts including their essential oils are becoming the63

emerging ingredients to fulfil part of the increasing demand for natural products with a64

green image.65

Essential oils (EOs), obtained from plant material including flowers, seeds, leaves,66

herbs, fruits and roots; are mainly used in cosmetic, pharmaceutical and in food flavours as a67

number of EOs has been registered for use as flavourings in foodstuffs (Burt 2004). More68

recently, studies have shown the potential applications of EOs, solely or in combination with69

other preservation processes to conserve food (Mahmoud et al., 2006; Solomakos et al.,70

2008); and a few food-preservatives containing EOs are already commercially available.71

It is well known that EOs possess antibacterial, antioxidant, antiviral and antimycotic72

properties as reviewed by several authors (Burt 2004; Fisher & Phillips, 2008). In cumin73

(Cuminum cyminum ) and laurel ( Laurus nobilis ) EOs, such activities were assigned to their74

phenolic and terpenic compounds includingcuminaldehyde, - pinene, ! -cymene, limonene,75

geranyl acetate, eugenol, " - pinene, and sabinene (Burt, 2004; Gachkar et al., 2007); and " -76

pinene, sabinene, -pinene, 1,8-cineole, linalool, terpinen-4-ol, " -terpineol and " -terpinyl77


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acetate (Bouzouita et al., 2001; Burt, 2004) respectively. The biological properties of cumin78

EOs were also attributed to other substances such as cuminal and cuminic alcohol (Oroojalian79

et al., 2010). In most cases, the preservative effects have been extensively studied using a80

direct-contact of antimicrobial assays, including different types of diffusion or dilution81

methods (Burt, 2004; Mejlholm, & Dalgaard, 2002). The direct applications of cumin/laurel82

EOs and investigation effects on fish fillets are rather scarce (Abou-Taleb et al., 2007; Erkan83

& Bilen, 2010; Erkan et al., 2010). 84

The production of wild and cultured sea breamSparus aurata in Mediterranean85

countries is playing a significant role in satisfying the increased demand for human86

consumption. However, there appears to be a stronger consumer preference for wild caught87

over cultured fish (Grigorakis, 2007). It is therefore of great interest to reinforce the88

acceptance of cultured-fish, and to increase its appeal by conducting comparative study on89

quality features and changes occurring in wild and cultured fish during post-harvesting90

procedures.91

To our knowledge, there are no studies in the literature on wild/farmed sea bream92

treated with cumin or laurel EOs. Therefore, the objectives of the present work were to93

determine the effect of the treatment with cumin or laurel EOs, as a natural preservative, on94

quality features of freshSparus aurata stored in ice and to compare this effect on wild/farmed95

specimens by assessing some physicochemical and microbiological parameters. The effect of96

EOs on some technological aspects was equally included by investigating parameters such as97

muscle liquid holding and protein properties.98

Materials and methods99

Fish raw materials 100

Fresh cultured gilthead sea bream (Sparus aurata ) (45 fishes with average weight and101

length: 19221 g and 23517 mm, respectively) were obtained in March 2007 from the102


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farming unit ''SCALA Pche Export'', located in Monastir (Tunisia). A culture land-based103

system and farm-made feeds (in the form of semi-moist pellets) were used for fish farming.104

Fish feed was prepared with fresh minced sardine (60%), fishmeal (20%), soybean oil cake105

(15%), wheat bran (3%), cod liver oil (0.5%), vitamin premix (1%) and mineral premix106

(0.5%) ingredients. Fish feed contained 55% protein, 19% fat, 13.5% carbohydrates, 11%107

ashes and 1.5% crud fibre as measured in dry matter. Fresh wild sea bream (45 fishes with108

average weight and length: 16932 g and 25425 mm, respectively) were caught by109

commercial longline fishing gear during the same day, from the coastal water of the same area110

(Monastir, Tunisia).111

Essential oil extraction112

Dried cumin (Cuminum cyminum ) seeds and laurel (or sweet bay) ( Laurus nobilis )113

leaves were purchased from a local retail spice market (Tunis, Tunisia). Essential oil (EO)114

was prepared by hydro-distillation of dried ground plant material (500g of each) for 3 hours115

using a Clevenger-type apparatus. The obtained EOs from each spice were dried separately116

using anhydrous sodium sulphate and stored in dark at 4C until required(Skandamis et al.,117

2000; Oroojalian et al., 2010).118

Preparation of fish samples and storage conditions 119

All fishes (wild/farmed) were killed by immersing in ice cold water and delivered120

(packed into an insulated polystyrene box with ice) to the laboratory, within 3 hours of121

harvesting. Upon arrival, fishes were immediately weighed, gutted, headed, washed, and122

filleted. Six farmed (F) and wild (W) fish fillets were immediately sampled (day 0),two 123

portions of tissues from each fillet were cut, one portion was served to microbiological124

analysis and the other deep immersed in liquid nitrogen and stored at -80C until125

proximate/biochemical analysis. Each EO was added to wild/farmed filleted samples in126

appropriate volume (0.5% v/w) to the surface (two sides) of each fillet using a pipette127


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followed by mild uniform distribution (directly with gloved fingers) of the oil for each128

sample. All filleted samples were vacuum-packaged at a local fish processing company129

(HDPM, Tunis, Tunisia) in food grade (EU Directive 2002/72/EC-2004/19/EC)130

polyethylene/polyamide pouches (16 x 25cm, 100m in thickness) provided by the same131

company, using a Multivac C550 packaging machine (Multivac, Wolfertschwenden,132

Germany). Packed fillets (3 fillets per pouch) were separated into six lots (30 fillets in each133

lot): wild and farmedfish fillets vacuum-packaged without EOs (WV and FV respectively);134

wild and farmed fish fillets vacuum-packaged after addition of 0.5% cumin oil (WVC and135

FVC, respectively);wild and farmed fish fillets vacuum-packaged after addition of 0.5%136

laurel oil (WVL andFVL, respectively). All packed fillets were immediately stored in ice137

(ratio of 1:1 (w/w)) into polystyrene boxes provided with holes for drainage. Boxes were138

covered and stored in a refrigerator (2-4C) for up to 20 days. Flesh sampling from each lot139

was performed on days 0, 5, 10, 15 and 20. All chemicals used in this work were obtained140

from Sigma-Aldrich-Fluka Company Ltd. (Poole, Dorset, UK), unless otherwise specified.141

Chemical analysis142

Crude fat was extracted using chloroform/methanol (2:1 v/v) solution according to Bligh143

& Dyer (1959). Total carbohydrates was analysed by using the colorimetric method of Dubois144

et al. (1956) with glucose as standard solution. Moisture content was determined by air-drying145

of a portion of minced fish fillet at 1032 C for 24 h (method 950.46), crude protein analysis 146

according to the Kjeldahl method using potassium sulphate and copper (II) sulphate as the147

catalysts (method 981.10) and ash by incineration in a muffle furnace at 550 2 C for 24 h148

(method 938.08), according to the official methods of AOAC (1995).149

Microbiological analysis150

Sea bream flesh (10g) obtained from each fillet, were transferred aseptically to a151

Stomacher bag (Seward, West Sussex, UK) containing 90 ml of sterile 0.1% peptone water152


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(Oxoid, Basingstoke, UK) and homogenized for 1 min using a laboratory blender Stomacher153

400 (Seward, West Sussex, UK) at high speed.Tenfold successive dilutions were made with154

0.1% peptone water from these homogenates as required. Bacterial counts were determined155

using plate count agar medium (Oxoid, Basingstoke, UK). The inoculated plates were156

incubated at 30C for 48 h and at 4C for 72 h for total viable mesophilic and psychrotrophic157

bacterial counts respectively (Harrigan & McCance, 1976). Microbiological counts were158

expressed as log colony forming units (cfu) per gram of sample.159

TVB-N, TMA-N and total free amino acids (NPS) contents160

Nitrogenous compounds were extracted from fish flesh as described inAttouchi &161

Sadok (2010).Analysis were performed according to Ruiz-Capillas & Horner (1999) for total162

volatile basic nitrogen (TVB-N), to Sadok et al. (1996) for trimethylamine (TMA-N) and to163

Sadok et al. (1995) for total free amino acids measured as ninhydrin-positive substances164

(NPS).165

Thiobarbituric acid (TBA) value166

TBA value was measured directly on homogenised flesh sample according to the167

method of Hamre et al. (2001). TBA values were quantified by reference to malondialdehyde168

(MDA) as standard solution prepared from 1,1- to 3,3-tetraethoxypropan. Absorption was169

measured at 532nm wave length with a Smart Spec-plus spectrophotometer (Bio-Rad,170

Hercules, CA) and data were expressed as mg MDA/ kg sample.171

Determination of pH172

Value of pH was determined, at room temperature, on homogenised fillet samples in173

distilled water (1:2 w/v) using a calibrated digital pH-meter (Inolab pH-720, WTW,174

Weilheim, Germany) according to AOAC process (method 981.12, AOAC, 1995).175

Liquid-holding capacity measurement176


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A centrifugation method, as described by Rr et al. (2003) was applied to evaluate the177

liquid-holding capacity (LHC) of sea bream fillets. A portion of frozen chopped muscle178

samples (2.0g) was placed in a centrifuge tube with a weighted (V1) filter paper (Whatman,179

Maidstone, England). Following muscle thawing, tubes were centrifuged (3K30 Sigma180

Centrifuge, Osterode am Harz, Germany) at 4000g for 10 min at 10C, and the wet paper was181

weighted (V2) before drying at 50C to constant weight (V3). The percentage value of liquid182

loss (LL) was calculated as 100x (V2-V1)/S, where S = weight of muscle sample, water loss183

as 100x (V2-V3)/S and fat loss as 100x (V3-V1)/S. All losses were expressed as percentage184

of muscle wet weight.185

Protein extraction and SDS-PAGE electrophoresis186

Muscle proteins were fractionated according to the procedure of Hashimoto et al.187

(1979). Samples were kept on ice throughout the process in order to prevent heating. Fish188

muscle (1g) was homogenized in 10ml of chilled phosphate buffer (15.6mM Na2HPO4,189

3.5mM KH2PO4, pH 7.5) and then centrifuged at 5000 g for 15 min at 4C. The pellet was re-190

suspended in 10ml of the same buffer and centrifuged as mentioned above. Supernatants of191

both consecutive centrifugations were mixed together to constitute the sarcoplasmic proteins192

fraction (SPP). Remaining pellets were again extracted twice with 10ml of chilled phosphate193

buffer (15.6mM Na2HPO4, 3.5mM KH2PO4, 0.45M KCl, pH 7.5) and centrifuged as194

previously described. Supernatants were collected as myofibrillar proteins fraction (MFP).195

The method of Bradford (1976) was used to quantify protein concentration in both fractions. 196

The obtained SPP and MFP fractions were stored at -80C until analysis. Sodium dodecyl197

sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of SPP and MFP fractions was198

performed according to Laemmli (1970) in a Mini Protean II electrophoresis system (Bio-199

Rad, Hercules, CA), using 5% polyacrylamide stacking gels and 15% separating gels. SPP200

and MFP extracts were diluted (4:1) in sample buffer (60 mM Tris-HCl, pH 6.8, 25%201


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glycerol, 2% SDS, 0.1% bromophenol blue, 14.4mM -mercaptoethanol) and boiled for 5202

min. Appropriate volumes were placed in each well in order to load ca. 30 and 35g proteins203

per well from SPP and MFP extracts, respectively. Protein molecular weight markers of 225,204

150, 100, 75, 50, 35, 25 and 10 kDa (Promega, Madisson, USA), were added (5l) to each205

gel. Electrophoresis was carried out at constant current of 30 mA per gel. Gels were then206

stained using Coomassie Brilliant Blue (R-250) method and analysed with a Digi-Doc-IT207

system (UVP, Upland, Ca). 208

Statistical analysis209

All experiments were carried out in duplicate on different occasions. Analyses were run210

in triplicate for each replicate and averaged for each fillet. Results were presented as mean 211

standard deviation. Data from the different measurements were subjected to ANOVA using212

SPSS version 11.01 software (SPSS Chicago, IL, USA). Tukeys test was used to check for213

significant differences among mean values at the 5% level. Data were also explored by214

principal component analysis (PCA) (Martens & Ns, 1989) using multivariate statistical215

software (The Unscrambler version 9.2, CAMO Software AS, Oslo, Norway). Leverage216

correction of all the data was applied. The variables were weighted with the inverse of the217

standard deviation of all the objects in order to compensate for the different scales of the218

variables.219

Results and discussion220

Proximate composition221

Proximate composition as a percentage of wet-weight fillets in fresh wild sea breamis222

given in Table 1. Results are similar to that reported in a previous work (Attouchi & Sadok,223

2010). Fat and carbohydrates contents were high (p


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fat level in the feed and to reduced activity of cultured fish. Additionally, the unlimited access227

to feed in intensive sea bream farming system leads to increased muscle carbohydrates228

(Kristoffersen et al., 2006). In contrast, no significant difference (p>0.05) in protein and ash229

contents were found between wild/farmed flesh fish lots. Similar results have been also230

obtained in other works, and protein content variation was reported to be mainly determined231

by species type, genetic characteristics and fish size (Grigorakis, 2007; Attouchi & Sadok,232

2010).233

During ice storage, the moisture and fat contents showed no significant changes (TABLE 2) 234

with mean values of 79.55 and 75.56% and 1.54 and 4.90% respectively for wild and cultured235

fish. Statistic analysis have shown that moisture and fat contents were not significantly236

influenced (p>0.05) by storage time nor by EOs treatment in all fish groups vacuum-packaged237

fillet. Similar results were found for wild/cultured sea bream when treated with powdered238

thyme (Attouchi & Sadok, 2010).239

Microbiological changes240

Changes in the microbial flora of all filleted fish samples during ice storage are241

illustrated in Table 3, representing a foremost comparative study for wild and cultured sea242

bream. Mesophilic bacterial counts (MBC) in fresh wild/farmed sea bream were respectively243

(3.220.53 and . 0. log cfu/g), while psychrotrophic bacterial counts (PBC) were244

respectively ( . 0. and .110.29 log cfu/g). Considering the proposed upper limit for245

aerobic plate count of 5x105 cfu/ g for fresh fish (ICMSF, 1986), MBC and PBC showed246

relatively low initial values in all samples, indicating the good quality of fish and that fish247

processing was done accurately. Initial microbial counts in both fish populations were found248

to be similar to those reported in cultured sea bream by Chouliara et al. (2004). Furthermore,249

no significant difference (p>0.05) was found in initial MBC and PBC values between250

wild/farmed sea bream lots.251


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MBC and PBC values exceeded the value of 7 log cfu/g (upper acceptability limit for277

freshwater and marine species; ICMSF, 1986) on day ca. 16 in FV-samples. Such value has278

never been reached in wild and EOs-treated samples up to 20-days storage period. Chouliara279

et al. (2004) reported that mesophilic counts of salted VP farmed sea bream fillets reached the280

value of 7 log cfu/g after 14 days storage at 4C.281

Changes in TVB-N and TMA-N282

Development of TVB-N values in all sea bream fillet lots are given in Table 4. At the283

beginning and throughout ice storage, WV-lot had significantly (p


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(Abou-Taleb et al., 2007). Indeed, this finding may be attributed to the inhibition of spoilage302

bacteria activity caused by the antibacterial properties of active compounds of these EOs as303

explained above. In consequence, the use ofcumin or laurel EOs combined with VP extended304

the shelf life of fish fillets by about 5 days in both wild/farmed sea bream.305

It is worth noting that the preservative effect ofcumin or laurel EOs was more prominent in306

wild than in farmed fish samples (Table 4). Such results may be explained by the diluting307

effect of the oily matrix of fish on the EOs phenolic/terpenic lipophilic components reducing308

their antibacterial effectiveness as has been reported by Burt (2004).309

Changes in TMA-N values in all samples during ice storage are exposed in Table 4. At310

day 0, low TMA-N contents with no significant (p>0.05) difference was recorded in fresh311

wild/farmed sea bream fillets, indicating good freshness of the product. These values were in312

the range of TMA-N levels noted in fresh sea bream in other studies (Goulas & Kontominas,313

2007, Attouchi & Sadok, 2010). Throughout the refrigerated storage, TMA-N levels increased314

exponentially with r 2 > 0.7 in all fish fillets. The TMA contents rose in the order WVL-315

WVC


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established and may be attributed to antibacterial properties of active compounds of the327

considered EOs as previously described. This finding matched also those acquired by Abou-328

Taleb et al. (2007). As showed in Table 4, the effect ofcumin or laurel EOs were more329

accentuated in wild than in farmed fish fillets, this fact may be explained by the dilution330

action of high fat content in farmed fish on active compounds of EOs (Burt, 2004).331

Changes in free amino-acids (NPS) and Thiobarbituric acid (TBA)332

Free amino acids are produced in fish as a result of muscle proteolysis, and as muscle333

spoilage progresses, these compounds serve as a substrate for microbial growth (Ruiz-334

Capillas & Moral, 2003). NPS values recorded during chilled storage of all sea bream samples335

are indicated in Table 5. Initially, mean NPS level of fresh farmed sea bream fillets was336

slightly higher than that of wild fish with no significant difference (p>0.05).337

Throughout ice storage, NPS values of WV and FV groups remained constant throughout338

storage, and showed significant increase at the end of this period. Ruiz-Capillas & Moral339

(2003) reported that levels of free amino acids in hake, packed under combined system of340

atmospheres, fluctuated or remained constant all over chilled storage. In the present study,341

EOs treated lots undergone a slight increase in NPS levels at day 5 and some fluctuations all342

over this period with no significant differences. The earlier rise of NPS in WVC, WVL, FVC343

and FVL samples concurred with their lowest microbiological counts, while the following344

fluctuations reflected the resumed bacteria growth as muscle spoilage progresses.345

Assessment of lipid oxidation was undertaken by TBA measurement in all VP fillets lots346

during storage (Table 5). Low initial TBA content was observed in fresh farmed/wild sea347

bream (0.41 and 0.25 mg MDA/kg respectively) with a significant difference (p


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polyunsaturated fatty acids in farmed fish (Grigorakis, 2007). While referring to previous352

TBA values obtained in aerobically ice storage wild/farmed sea bream (Attouchi & Sadok,353

2010), the use of VP alone or in combination withcumin or laurel EOs played a key role in354

keeping TBA values at low levels in all fillet samples throughout the entire storage period.355

Moreover, TBA content of laurel treated under VP sample (particularly WVL), was extremely356

low. In literature, it has been stated that use of the whole cumin seeds and laurel leaves or357

their extracts, possessing strong antioxidant activity, (Burt, 2004; Gachkar et al., 2007) can358

control lipid oxidation in muscle food such as mullet fish (Abou-Taleb et al., 2007) and359

frozen chub mackerel (Erkan & Bilen, 2010). The antioxidant activity of bothcumin and360

laurel EOs was related to their phenolic, terpenic as well as other compounds as cited above.361

According to Connell (1990), TBA levels of 1-2 mg MDA/kg of fish flesh are generally362

considered as the limit beyond which fish will normally develop off-flavours and off-odours.363

The TBA contents of the present sea bream fillet samples exceeded the value of 1 mg364

MDA/kg after ca. 15 and 18 days of storage period for (FV) and (WV) groups respectively,365

whilst WVC, WVL, FVC and FVL-groups never reached this limit value throughout the366

whole storage period.367

Changes in pH, LHC, WHC and Fat loss368

Assessment of pH values in all fish samples during ice storage are displayed in Table 6.369

A significant difference (p


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subsequently to reduce muscle post-mortem pH in response to anaerobic degradation of377

glycogen (Kristoffersen et al., 2006). Over the storage period, pH value undergone different378

change patterns in sea bream batches. In WV-lot, pH showed a significant rise toward the end379

of storage time, whereas pH values of WVC and WVL-lots remained unaffected during this380

period. As reported in literature, the increase of pH values during storage was attributed to the381

production of basic compounds such as ammonia, trimethylamine and other biogenic amines382

by fish spoilage bacteria (Kyrana et al., 1997). In this study, pH values of wild sea bream383

fillets (WV, WVC and WVL) demonstrated a good correlation with TVB-N and TMA-N384

changes. In addition, pH stability observed in WVC and WVL-samples may be due to EOs385

inhibitory effects on microbial growth, which in turn, delay the formation of basic nitrogen386

compounds.387

In FV-lot, pH values showed a significant increase only after 10 days of storage which388

coincided with microbiological loads rise, flowed by a decrease toward the end of this period.389

This observation may be related to the growth of lactic acid bacteria (LAB) as suggested by390

numerous reports. Indeed, LAB bacteria are facultative anaerobic species, naturally present in391

relatively large number in vacuum-packaged farmed sea bream (Chouliara et al., 2004) as392

well as in farmed cod (Olsson et al., 2007). Such bacteria may contribute to unchanged or393

declined muscle pH, primarily by producing lactic acid which acts to buffer basic metabolites,394

and secondly by producing bacteriocin which acts to inhibit growth of other kind of bacteria395

(Herland et al., 2007). As shown in Table 6, pH values undergone a preliminary increase at396

day 5 in FVC-group and remained unchanged toward the end of storage period, which was the397

case of FVL-lot all over this period. Furthermore, it is worth noting that despite its relatively398

higher TVB-N and TMA-N levels (see Table 4), final pH values recorded in FVC and FVL-399

lot were significantly (p


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tolerance toward the action of EOs due to their ability to generate ATP and to deal with402

conditions of osmotic stress (Burt, 2004).403

It is well known that determination of liquid-holding capacity (LHC) in meat and fish is404

important for economical reasons (weight decrease due to water loss) and for sensory405

properties (colour, juiciness and tenderness) (Olsson et al., 2003a).Variation of fat loss (FL),406

water loss (WL) and liquid loss (LL) levels in different treatments are included in Table 6.407

Initially and throughout ice-storage, the FL in wild fillets were significantly (p


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WV and FV-lots respectively, whereas in an earlier study (Attouchi & Sadok, 2010), LL427

values of wild/farmed sea bream fillets increased significantly after 7 days of aerobic ice428

storage, suggesting that application of vacuum-packaging may delay LL changes in fish429

fillets.430

However, when fish fillets were treated with EOs, significant LL increase (p


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and hereby quality characteristics such as LHC. Moreover, growth of some specific spoilage452

bacteria and interaction between them can be implicated in fish muscle-LHC variation453

(Olsson et al., 2007).454

Electrophoretic (SDS-PAGE) analysis455

Changes in electrophoretic profiles of SPP and MFP extracts of all sea bream treatments456

are shown in Fig.1. SPP extracts of wild and farmed fillets lots (Fig. 1a-b respectively)457

contained 11 principal bands after SDS-PAGE separation, with relative molecular weight of458

100, 62, 50, 41, 36, 33, 26, 24, 22, 10 and 9 KDa. Several fractions were obtained for MFP459

extracts of wild and farmed fillets lots (Fig. 1c-d respectively), although the most contributing460

to total protein content, in terms of optical density, were the bands of 200, 108, 42, 32, 18 and461

16 KDa. According to Delbarre-Ladrat et al. (2006) and several other studies, these bands462

were tentatively identified as myosin heavy chain (MHC), -actinin ( -ACN), actin (AC),463

tropomyosin (TMP) and myosin light chains (MLC) respectively. At first sight, comparison of464

SPP and MFP electrophoretic patterns among fresh wild/farmed fish did not show evident465

differences between the two fish types. In general terms, no substantial changes were466

observed in the SSP and MFP extracts profiles of all sea bream lots as a consequence of467

treatments and of storage progression. However, minor changes were mainly noted in low468

molecular weight protein bands in both fractions. For instance, electrophoregram of SSP469

extract showed the appearance of 23, 18 and 12 KDa and the decrease of 16KDa band density470

from the day 10 of ice storage in WV group, whereas these polypeptides were revealed in471

addition to a 14 KDa band from the day 5 of storage period in WVC and WVL groups (Fig.472

1a). In FV lot, SSP electrophoresis profile showed the appearance of 14 and 12 KDa bands473

from the day 10 of ice storage, and the increase of 18KDa band density at the end of this474

period, while these bands were observed from the day 5 of storage period in FVC and FVL475

groups with different densities (Fig. 1b). MFP electrophoretic pattern indicated the occurrence476


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of low density 22KDa band in WV and WVL lots from the day 0 and 5 respectively, the477

concentration of this polypeptide seemed to be higher throughout the entire experiment in478

WVC lot (Fig. 1c). Additionally, a continuous increase of 26 and 24 KDa band densities was479

observed from day 0 and from day 5 respectively in FV-lot until the end of storage. However,480

these bands were occurred with lower intensity from the day 5 to the end of storage period in481

FVC and FVL-groups (Fig. 1d). In most cases, the major reported protein changes during482

storage are weakening of the Z-line, degradation of titin, nebulin, dystrophin, desmin, as well483

as release of -actinin from the Z-line, and breakdown of collagen junctions between484

myotomes (Delbarre-Ladrat et al., 2006). However, the current study divulged small changes485

in 26-14 KDa area in both SSP and MFP fraction that was relatively concurred with bacterial486

load rise (day 10) only in WV and FV lots. Furthermore, it was clear that addingcumin or487

laurel EOs to VP sea bream was speculated to enhance protein degradation. Given the488

antibacterial property of the considered EOs, this degradation seemed to be caused by489

endogenous enzymes. Calpain proteases were suggested to be responsible of such proteolysis490

based on their early post-mortem activation and on their activity enhancement by reducing491

atmosphere combined with antioxidant treatment. For instance, since desmin (a protein of 55492

KDa) was known as calpain substrate, activation of calpain might explain breakdown of this493

myofibrillar protein which, in turn, may influence muscle-LHC (Huff-Lonergan et al., 2005).494

In the present work, better information on the presence of such specific proteins and their495

degradation as a consequence of treatment/storage can be provided by the use of more496

sensitive techniques based on SDS-PAGE such as immunoblotting study.497

Principal component analysis (PCA)498

With the aim to better understand which factors mostly affect the quality of sea bream499

batches and to check whether there were correlations between muscle quality parameters, EOs500

treatment and storage time, a PCA model was undertaken on a matrix with 180 objects (sea501


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bream fillets) and 12 variables (pH, TBA, NPS, TMA-N, TVB-N, LL, PBC, MBC, cumin or502

laurel EOs treatment, origin (wild or farmed) and storage time). The fat and moisture contents503

were not included in the model since they would dominate separation between wild/farmed504

groups. It was found that two principal components (PCs) explained 60% of the variations in505

the data set. The scores and loadings of PC1 and PC2, representing 42% and 18% of the total506

variation, are given in Fig.2. The score plot shown in Fig.2a displayed a clear distinction507

between wild/farmed sea bream irrespective of fillet treatments. Similar results were recorded508

for wild/farmed sea bream (Attouchi & Sadok, 2010) and for wild/fed cod (Kristoffersen et509

al., 2006). The discrimination was principally along the principal component two (PC2),510

where the farmed samples are located in the upper of the score plot and the wild ones in the511

lower area.512

The loadings plot (Fig. 2b) shows that the variations in muscle pH are mainly described513

by PC2 and correlated to whether the fish is wild or farmed. As shown in Fig. 2b, PC1 is514

spanned out by the parameters laurel EOs treatment on the left hand and TVB-N, TMA-N,515

TBA, PBC, MBC and storage time on the right hand sides. Thus, sample with advanced time516

storage are associated with high level of TVB-N, TMA-N, TBA, PBC and MBC517

independently of fish origin, while sample treated with laurel EOs are associated with low518

level of these parameters. Cumin EOs treatment appeared to own weaker effect on all519

considered parameters since it was positioned near the intersection point of the two PCs axes.520

In addition, laurel EOs treatment and storage time factors describe a lower influence on LL521

and NPS attributes than that on other measured attributes. From the loading plot it is seen that522

variations in LL and NPS (free amino-acids) are described both in PC1 and at lower rank in523

PC2 and thus both dependent on type of fish and time of storage. Moreover, LL variation was524

strongly correlated to free amino-acids (NPS) production and consecutively to proteolysis525


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activity in all fillet samples. Data from PCA analysis confirmed all acquired results as526

previously explained.527

Conclusions528

Laurel and at a lesser extend cumin EOs exhibited a preservation effects on VP-sea529

bream fillets during ice storage. Independently of fish origin, lower level of TVB-N, TMA-N,530

TBA, PBC and MBC were found in sea bream fillets treated with 0.5% EOs. Thus the531

treatment with laurel or cumin EOs extended the shelf life of fish fillets by approximately 5532

days in both wild and farmed sea bream compared to non-treated lots. However, EOs533

application seemed to enhance endogenous protease activation, inducing increased muscle534

proteolysis and in turn a drop in muscle-LHC especially in cultured fish. Such results were535

confirmed by PCA analysis. The actions of laurel and cumin EOs on structural proteins, as536

well as their synergistic effects were not identified in this study; further investigations on537

these mechanisms are needed as it would provide insights that may be useful from a538

technological point of view.539

Acknowledgements540

This study was supported by the Tunisian Ministry of Higher Education and Scientific541

Research (Project N: 25/TG/05).542

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Olsson G B, Olsen R L & Ofstad R (2003 a) Post-mortem structural characteristics and water-636

holding capacity in Atlantic halibut muscle. LWT-Food Science and Technology , 36, 125-637

133.638

Olsson G B, Olsen R L, Carlehog M & Ofstad R (2003 b) Seasonal variation in chemical and639

sensory characteristics of farmed and wild Atlantic halibut ( Hippoglossus hippoglossus ).640

Aquaculture , 217, 191-205.641

Olsson G B, Seppola M A & Olsen R L (2007) Water-holding capacity of wild and farmed642

cod (Gadus morhua ) and haddock ( Melanogrammus aeglefinus ) muscle during ice643

storage. LWT-Food Science and Technology , 40, 793-799.644

Oroojalian F, Kasra-Kermanshahi R, Azizi M & Bassami M R (2010) Phytochemical645

composition of the essential oils from three Apiaceae species and their antibacterial646

effects on food-borne pathogens. Food Chemistry , 120, 765-770.647


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Rr A M B, Regost C & Lampe J (2003) Liquid holding capacity, texture and fatty acid648

profile of smoked fillets of Atlantic salmon fed diet containing fish oil or soybean oil.649

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Figure captions673

674

Figure 1: SDS-PAGE patterns of sarcoplasmic (a-b ) and myofibrillar (c-d ) proteins extracted675

respectively from vacuum-packaged wild and farmedSparus aurata fillets treatments after 0;676

5; 10; 15 and 20 days of ice storage677

678

(WV): vacuum-packaged (VP) wild sea bream fillets; (WVC): VP wild sea bream fillets treated with 0.5%679

cumin EO; (WVL): VP wild sea bream fillets treated with 0.5% laurel EO; (FV): VP farmed sea bream fillets;680

(FVC): VP farmed sea bream fillets treated with 0.5% cumin EO; (FVL): VP farmed sea bream fillets treated681

with 0.5% laurel EO. The numbers above the lanes indicate the days of ice storage. Arrows on the gels indicate682

the most changed bands throughout storage. M: Protein molecular weight markers; MHC: myosin heavy chain;683

-ACN: alpha- actinin; AC: actin; TMP: tropomyosin and MLC: myosin light chain 1and 2.684

685

686

Figure 2: Score (a ) and loading (b ) plots of the principal component analysis model carried687

out on all X-variables (pH, TBA, NPS, TMA-N and TVB-N, MBC, PBC, LL, cumin EO688

treatment, laurel EO treatment, origin (farmed (F) or wild (w)) and storage time).689

690

PC1 and PC2 explained 42% and 18% of the variations of the data set respectively. Wild and farmed groups are691

encircled.692

693694

695

696


29/37

ure


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Figure 2

gure


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1

2

3

4

5

6

7

Table 1: Proximate composition of fresh wild and farmedSparus aurata 8

Wild FarmedMoisture A 79.171.01a 75.101.03

Protein B 19.041.02a 19.950.91a

Total lipid 1.530.27a 4.820.44

Ash 1.360.10a 1.420.11a

Carbohydrate 0.22 0.03 a 0.32 0.03 9

A-E g/100g wet fillet; Data are mean standard deviation, n = 6; Means within the same row with different10

superscript are significantly different (p< 0.05) 11

12

13

14

1516

17

18

19

20

21

ble


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Table 2: Changes in the moisture and total fat of each sea bream fillet groups during storage22

period 23

Days Analysis Group

WV

Group

WVC

Group

WVL

Group

FV

Group

FVC

Group

FVL

0 Moisture

Total fat B

79.171.01a

1.530.27a

79.171.01a

1.530.27a

79.171.01a

1.530.27a

75.101.03

4.820.44 b

75.101.03

4.820.44 b

75.101.03

4.820.44 b

5 Moisture

Total fat

79.710.74a

1.680.35a

81.150.69a

1.380.22a

79.931.81a

1.430.21a

76.770.75a

4.980.45a

74.931.34a

4.950.55 a

76.071.31a

4.880.37 a

10 Moisture

Total fat

80.440.61a

1.520.38 a

77.450.48a

1.430.36 a

77.640.70a

1.570.23 a

75.351.31a

4.830.59 a

74.171.38a

5.120.61 a

73.531.64a

4.720.68 a

15 Moisture

Total fat

80.600.48a

1.670.22 a

79.700.57a

1.420.41 a

78.040.69a

1.780.43 a

75.180.90a

4.580.46 a

75.110.91a

4.820.68 a

75.611.80a

5.050.66 a

20 Moisture

Total fat

80.580.64a

1.420.15 a

81.090.74a

1.570.19 a

78.280.87a

1.630.20 a

76.571.73a

5.380.60 a

76.211.12a

4.450.60 a

77.161.13a

4.980.39 a

WV: vacuum-packaged wild sea bream fillets; WVC: vacuum-packaged wild sea bream fillets treated with 0.5%24

cumin EO; WVL: vacuum-packaged wild sea bream fillets treated with 0.5% laurel EO; FV: vacuum-packaged25

farmed sea bream fillets; FVC: vacuum-packaged farmed sea bream fillets treated with 0.5% cumin EO; FVL:26

vacuum-packaged farmed sea bream fillets treated with 0.5% laurel EO. Data are mean standard deviation, n =27

6; Means within the same row with different superscript are significantly different (p< 0.05).28A-B g/100g wet fillet29

30

31

32

33

34


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Table 3: Changes in mesophilic (MBC) and psychrotrophic (PBC) bacteria counts in each sea35

bream fillet groups during the storage period36

Days Analysis Group

WV

Group

WVC

Groupe

WVL

Group

FV

Group

FVC

Group

FVL0 MBC A

PBC B

3.220.53a

3.550.42a

3.220.53a

3.550.42a

3.220.53a

3.550.42a

2.830.26a

3.110.29a

2.830.26a

3.110.29a

2.830.26a

3.110.29a

5 MBC

PBC

3.360.66a

4.140.38a

ND

ND

2.420.24

ND

2.800.17a

3.290.30 b

2.770.28a

ND

ND

2.350.15c

10 MBC

PBC

4.310.75a

4.970.12a

3.150.50ac

3.850.15 bc

3.330.51ac

4.430.34ac

4.580.03

4.590.12a

4.240.06a

3.620.38 b

2.650.16c

3.840.33 bc

15 MBC

PBC

6.330.08ac

6.420.20a

5.350.10

5.430.18 b

5.770.24a

5.490.02 b

6.800.31c

6.640.14a

5.310.32

5.270.36 b

5.200.36

5.470.13 b

20 MBC

PBC

6.730.19a

6.720.23a

6.590.11a

6.200.10 b

6.720.02a

6.300.16ab

7.240.03a

7.620.14c

6.300.33

6.160.21 b

6.380.54

6.130.20 b 37




with 0.5% laurel EO. Data are mean standard deviation, n = 6; Means within the same row with different41

superscript are significantly different (p< 0.05). ND: not detected.42A-B log cfu/g43

4445

46

47

48

49

50


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51

Table 4: TVB-N and TMA-N changes of each fillet groups during the storage period52

Days Analysis Group

WV

Group

WVC

Group

WVL

Group

FV

Group

FVC

Group

FVL0 TVB-N

A

TMA-N B

11.070.85a

0.230.05a

11.070.85a

0.230.05a

11.070.85a

0.230.05a

13.200.74

0.170.03a

13.200.74

0.170.03a

13.200.74

0.170.03a

5 TVB-N

TMA-N

13.872.20a

0.160.02a

13.490.65a

0.750.04 b

13.491.91a

0.490.04c

19.900.68

0.130.02a

16.950.70c

0.420.05d

17.281.46c

0.430.02d

10 TVB-N

TMA-N

14.142.44ac

0.250.01a

14.151.62ac

0.920.06 b

12.670.59a

0.540.02cd

21.421.73

0.680.12e

19.060.38

0.590.04ce

16.080.81c

0.440.02d

15 TVB-N

TMA-N

24.591.26a

1.810.17a

15.630.90

1.090.07 b

15.141.77

0.630.04c

33.772.01c

2.960.19d

21.040.89a

1.260.10 b

20.022.12a

0.840.08e

20 TVB-N

TMA-N

35.801.59a

4.420.29a

19.870.66

1.060.10 be

18.741.47

0.750.03 b

41.432.06c

3.250.20c

27.670.52

2.770.24d

32.011.39e

1.220.09e

53





superscript are significantly different (p< 0.05).A-B: mg N/100g58

59

60

61

62

63

64

65


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66

Table 5: NPS and TBA changes of each fillet groups during the storage period67

Days Analysis Group

WV

Group

WVC

Group

WVL

Group

FV

Group

FVC

Group

FVL0 NPS A

TBA B

0.930.38a

0.250.02a

0.930.38a

0.250.02a

0.930.38a

0.250.02a

1.240.37a

0.410.07 b

1.240.37a

0.410.07 b

1.240.37a

0.410.07 b

5 NPS

TBA

0.930.29a

0.560.11ac

1.630.40

0.420.04ab

1.360.36a

0.280.05 b

1.120.27a

0.620.14c

1.510.38a

0.450.09a

1.550.35a

0.480.07ac

10 NPS

TBA

0.890.27a

0.840.09a

1.130.33a

0.770.07a

0.960.19a

0.210.02 b

1.020.28a

0.710.07ac

1.430.31

0.590.12c

1.170.15a

0.610.03c

15 NPS

TBA

0.980.16a

0.910.06a

0.880.20a

0.710.09 be

0.900.30a

0.360.04c

1.130.43a

1.050.09d

1.310.34a

0.800.06ab

1.250.26a

0.630.05e

20 NPS

TBA

1.550.41a

1.390.09a

1.460.44a

0.890.10 b

1.010.30a

0.810.12 b

1.870.24

1.480.12a

1.250.26a

0.930.08 b

1.140.37a

0.850.11 b 68





superscript are significantly different (p< 0.05).A mmol AA/100g;B mg MDA/Kg73

74

75

76

77

78

79

80


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Table 6: Liquid loss (LL), water loss (WL), fat loss (FL) and pH changes of each sea bream81

fillet groups during the storage period82

Days Analysis Group

WV

Group

WVC

Group

WVL

Group

FV

Group

FVC

Group

FVL0 pH

LL A

WL B

FL C

6.650.03a

15.071.19a

13.690.95a

1.380.38a

6.650.03a

15.071.19a

13.690.95a

1.380.38a

6.650.03a

15.071.19a

13.690.95a

1.380.38a

6.220.02

18.750.71 b

15.890.73 b

2.860.28 b

6.220.02

18.750.71 b

15.890.73 b

2.860.28 b

6.220.02

18.750.71 b

15.890.73 b

2.860.28 b

5 pH

LL

WL

FL

6.740.02a

16.150.95a

14.770.79a

1.380.17a

6.660.01

24.211.54 b

22.651.41 b

1.560.18 b

6.580.01c

22.600.73 bc

21.170.73 bd

1.430.10a

6.280.02

20.321.73c

17.661.54c

2.650.29 b

6.410.01e

23.160.88 b

19.280.81cd

3.880.60c

6.300.01

24.311.76 b

20.601.70 bd

3.710.43c

10 pH

LL

WL

FL

6.650.02a

16.331.15a

14.970.93a

1.360.33a

6.620.01

23.561.61 bd

21.981.44 bc

1.580.35a

6.610.02

23.521.26 bd

22.091.12 b

1.430.32a

6.420.02c

19.250.93c

16.430.78ad

2.820.35 b

6.430.01c

24.251.49 b

19.841.54ce

4.410.48c

6.260.01

21.741.58d

18.201.49de

3.540.27d

15 pH

LL

WL

FL

6.710.02a

19.541.93a

18.221.76ad

1.320.19a

6.640.01

25.091.36 b

23.531.33 b

1.560.23a

6.680.03c

24.141.06 b

22.710.93 bc

1.440.23a

6.330.02

19.741.95a

16.671.65a

3.070.40 b

6.440.01e

25.671.44 b

21.961.29 bc

3.710.40c

6.310.01

24.231.46 b

20.771.68cd

3.460.31 bc

20 pH

LL

WL

FL

6.940.02a

19.761.71a

18.431.62a

1.340.12a

6.670.01

27.281.30 b

25.811.12 b

1.470.22a

6.630.01c

23.461.42c

22.180.97cd

1.280.56a

6.360.01

22.220.81ac

18.700.88a

3.520.46 b

6.530.01e

27.251.32 b

23.291.29c

3.960.37 b

6.360.02

23.871.95c

20.541.94ad

3.330.62 b 83




37/37



superscript are significantly different (p< 0.05).A-C (g/100g wet fillet)88

8990

91

92

93

Documents

A the Effects of Essential Oils Addition