H NMR spectroscopy MVA analysis PCA PLS-DA

Food Chemistry 196 (2016) 601–609

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H NMR metabolomic profiling of the blue crab (Callinectes sapidus) from the Adriatic Sea (SE Italy): A comparison with warty crab (Eriphia verrucosa), and edible crab (Cancer pagurus) Maurizio Zotti ⇑, Sandra Angelica De Pascali, Laura Del Coco, Danilo Migoni, Leonardo Carrozzo, Giorgio Mancinelli, Francesco Paolo Fanizzi ⇑ Department of Biological and Environmental Sciences and Technologies, Di.S.Te.Ba, University of Salento, Prov.le Lecce-Monteroni, Lecce 73100, Italy

a r t i c l e

i n f o

Article history: Received 5 March 2015 Received in revised form 7 September 2015 Accepted 24 September 2015 Available online 28 September 2015 Chemical compounds studied in this article: Omega-3 Fatty Acids (PubChem CID: 56842239) Cholesterol (PubChem CID: 5997) Phosphatidylcholine (PubChem CID: 45266626) Glutamate (PubChem CID: 33032) Homarine (PubChem CID: 3620) Betaine (PubChem CID: 247) Taurine (PubChem CID: 1123) Lactate (PubChem CID: 612) Glycine (PubChem CID: 750) Alanine (PubChem CID: 602)

a b s t r a c t The metabolomic profile of blue crab (Callinectes sapidus) captured in the Acquatina lagoon (SE Italy) was compared to an autochthonous (Eriphia verrucosa) and to a commercial crab species (Cancer pagurus). Both lipid and aqueous extracts of raw claw muscle were analyzed by 1H NMR spectroscopy and MVA (multivariate data analysis). Aqueous extracts were characterized by a higher inter-specific discriminating power compared to lipid fractions. Specifically, higher levels of glutamate, alanine and glycine characterized the aqueous extract of C. sapidus, while homarine, lactate, betaine and taurine characterized E. verrucosa and C. pagurus. On the other hand, only the signals of monounsaturated fatty acids distinguished the lipid profiles of the three crab species. These results support the commercial exploitation and the integration of the blue crab in human diet of European countries as an healthy and valuable seafood. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Callinectes sapidus Eriphia verrucosa Cancer pagurus 1 H NMR spectroscopy MVA analysis PCA PLS-DA

1. Introduction Marine crustaceans are widely used as food and feed supplement and provide an important contribution to capture fisheries and aquaculture worldwide (Stentiford et al., 2012). In addition,

⇑ Corresponding authors. E-mail addresses: [email protected] (M. Zotti), fp.fanizzi@ unisalento.it (F.P. Fanizzi). http://dx.doi.org/10.1016/j.foodchem.2015.09.087 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

the growing global demand for seafood has to face a declining phase of wild finfish availability and an increase in the importance of crustacean fisheries in the next decades is expected (Anderson, Flemming, Watson, & Lotze, 2011). Among others, crabs are an important shell fishery product, representing about 20% worldwide, approximately 21.8% in European waters and 5.3% in Mediterranean countries of all crustaceans caught and farmed (FAO, 2012). An outstanding example is represented by the Atlantic blue crab Callinectes sapidus (Rathbun, 1896), which in native areas – i.e., north-western coasts

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of the Atlantic ocean – supports important fisheries and contribute to sustain the economy of coastal areas. At the beginning of the twentieth century the species was recorded in European waters, probably introduced by ballast waters (Nehring, 2011). In the Mediterranean Sea it was introduced deliberately in the Aegean Sea in the ’30 (Artüz, 1990). To date the blue crab has been recorded in the Adriatic (Dulcˇic´, Tutman, Matic´-Skoko, & Glamuzina, 2011) and Ionian Seas (Carrozzo et al., 2014; Mancinelli, Carrozzo, Marini, Costantini et al., 2013; Mancinelli, Carrozzo, Marini, Pagliara, & Pinna, 2013), where established and potentially exploitable populations, comparable to those occurring in Aegean waters, have been identified. Available information on the quality of C. sapidus as seafood in the Mediterranean Sea are limited to Turkish populations and, so far, are restricted to the analysis of proximate composition and elemental contents (Küçükgülmez, Celik, Yanar, Ersoy, & Çikrikçi, 2006; Türkmen, Türkmen, Tepe, Mazlum, & Oymael, 2006). Indeed, in past decades investigations in the nutraceutical and functional food field have mainly relied upon a relatively limited series of standard chemical and physico-mechanical measurements of food quality. To date, the field is experiencing a rapid paradigm shift in academic, commercial and government sectors, due to the development of high-throughput, -omics technologies, in which a large (or even exhaustive) number of measurements can be taken in a relatively short time period (Bagchi, Lau, & Bagchi, 2010). Metabolomics is one of the four major types of omics measurements (together with genomics, transcriptomics, and proteomics), and is defined as the comprehensive, simultaneous determination of endogenous metabolites (i.e., small-molecular-weight molecules that link processes in cells, tissues and organisms to each other) at the molecular level and their global and dynamic changes over time in complex multi-cellular systems (Larive, 2007). Metabolomics have been proven to provide in-depth insights into the biochemistry of diets, toxicity, medicine, physiology and pathology (Hu & Xu, 2013). Specifically, in food chemistry metabolomic analyses are currently viewed as crucial procedures in the implementation of a ‘‘foodomics approach” investigating the food domain in parallel with the nutrition domain to optimize human health and wellbeing (Capozzi & Bordoni, 2013). Over the past ten years, nuclear magnetic resonance (NMR) spectroscopy, used in combination with multivariate data analyses, has been indicated as an effective analytical tool that can provide comprehensive information on the metabolic profiles in both vegetal and animal matrices (e.g., De Pascali et al., 2014; Del Coco et al., 2009, 2014). Indeed, such analytical approach is acknowledged as accurate, rapid and reliable for assessing seafood quality (Heude, Lemasson, Elbayed, & Piotto, 2014). Nevertheless, scant efforts have been made for crustacean seafood. The available examples of NMR application regard mainly non fresh food such as crab paste or thawed and cooked shrimp meat (Carneiro et al., 2013; Ye, An, Li, Mu, & Wang, 2014). NMR studies have also been used for the assessment of pathological and stress states of crustaceans (Schock et al., 2010). The general aim of the present study is to provide an advanced resolution of the quality of C. sapidus as a shellfish food using a foodomics approach based on metabolomics. Specifically, in order to estimate the nutritional value of C. sapidus found in the Salento Peninsula (SE Italy), an NMR-based approach was applied to investigate its metabolomic profile with respect to two marketed and exploited species in Italy, i.e., the autochthonous warty crab Eriphia verrucosa and the imported edible crab Cancer pagurus. E. verrucosa has a potential commercial

value in Mediterranean countries (Altinelataman & Dincer, 2007) and occurs episodically in Italian fish markets. So far scarce studies are available on this latter species and only for Black Sea populations (Altinelataman & Dincer, 2007; Ozogul et al., 2013). In contrast, the edible crab C. pagurus is particularly appreciated in Southern European countries, being imported throughout Europe mostly from Britain, Ireland, Norway, Sweden, Spain, France and Portugal (Barrento, Marques, Pedro, Vaz-Pires, & Nunes, 2008). For the first time 1H NMR spectroscopy and MVA (multivariate data analysis) were performed on the aqueous and lipid extracts of raw claw muscle with the aim to provide a comparison of the three edible species. Our results may encourage both integration in human dietary regime and exploitation in the Mediterranean shell fishery of the blue crab. This may contribute to control its ecological invasiveness and counteract overexploitation of autochthonous species with economic relevance. 2. Materials and methods 2.1. Sample collection C. sapidus and E. verrucosa specimens were captured in June 2014 in the Acquatina lagoon (SE Italy) using crab traps (60  60  60 cm) constructed of vinyl-coated 2  2 cm mesh wire with an upper and lower chamber. After collection, specimens were transferred alive in refrigerated containers to the laboratory, where they were sexed; for each crab species, five E. verrucosa males and eight C. sapidus males were randomly chosen, and their carapace width (in mm) and wet weight (in g) were determined. After measurements, crabs were euthanized by thermal shock ( 20 °C for 10 min). Simultaneously, five C. pagurus, originally captured along the French coasts of the English Channel (Bretagne), were purchased alive from a local fish market. After transfer to the laboratory under refrigerated conditions, crabs were measured and euthanized. 5.0 g of muscle tissue from each collected specimen were taken by hand from both claws using a scalpel. They were frozen ( 20°C) and used for subsequent NMR analyses. 2.2. NMR spectroscopy The lipid and aqueous extracts from the claw muscles were obtained by using Bligh-Dyer method (Bligh & Dyer, 1959). The aqueous extracts were dissolved in D2O containing buffer (potassium phosphate buffer pH 7.4, 4% NaN3, 0.08% TSP) and mixed. 600 lL of the supernatant were placed in a 5 mm outer diameter NMR tube. The lipid extracts were dissolved in 600 lL of CDCl3 containing 0.03% vol/vol TMS and placed in a 5 mm outer diameter NMR tube. NMR measurements were performed on a Bruker Avance III NMR spectrometer (Bruker Corporation, Karlsruhe, Germany) operating at 400.13 MHz (1H observation), equipped with a BBI 5 mm indirect detection probe incorporating a z axis gradient coil and automatic tuning-matching (ATM). NMR spectra were acquired using Topspin 2.1 (Bruker, Biospin). Automated tuning and matching, locking and shimming using the standard Bruker routines ATMA, LOCK, and TopShim were used to optimize the NMR conditions. A time delay of 5 min was set between sample injection and pre-acquisition calibrations to ensure complete temperature equilibration (300 K). For aqueous extracts, one-dimensional spectra with a transverse- relaxation-filter incorporating pulse sequence (referred to as Carr–Purcell–Meiboom–Gill spin-echo sequence, CMPG)

M. Zotti et al. / Food Chemistry 196 (2016) 601–609

(cpmgpr1d) including water signal saturation were acquired. For each sample 128 free induction decays (FIDs) were recorded using a spectral width of 6009.615 Hz, 32k data points, an acquisition time of 2.73 s, a relaxation delay of 5 s, and a mixing time of 100 ms. All aqueous extracts spectra were referenced to the TSP signal (d = 0.00 ppm). For lipid extracts, 1D1H ZG spectra were acquired using 64 FIDs, 32K data points, a spectral width of 6009.615 Hz, an acquisition time of 2.73 s and a relaxation delay of 2 s. Lipid extracts spectra were referred to TMS signal (d = 0.00 ppm). For both aqueous and lipid extracts, the FIDs of 1D spectra were multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz before Fourier transformation, phasing, and baseline correction. Metabolites were assigned on the basis of 2D NMR spectra analysis (2D 1H J res, 1H COSY, 1H 13C HSQC and HMBC) and comparison with published data (Mannina et al., 2008; Schock et al., 2010; Stabili et al., 2014; Tikunov, Johnson, Lee, Stoskopf, & Macdonald, 2010; Ye, Zhang, Tang, & Yan, 2012; Ye et al., 2014).

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and indicates goodness of the fit. The Q2 parameter is defined as the proportion of variance in the data predictable by the model and indicates predictability, which is extracted according to the internal cross-validation default method of SIMCA-P software. For each PLS-DA model built, a combination of the loading scores, variable influence on projection (VIP) parameters and p(corr) was examined to identify which metabolites most contributed to the data clustering. Loading scores describe the correlations between the original variables and the new component variables. VIP parameters are essentially a measure of the extent to which a particular variable explains the Y variance (class membership) and p(corr) represents the loadings scaled as a correlation coefficient (ranging from 1.0 to 1.0) between the model and original data. (Wheelock & Wheelock, 2013).

3. Results and discussion 3.1. NMR spectroscopy

2.3. NMR data reduction and preprocessing NMR spectra were processed using Topspin 2.1 (Bruker, Biospin) and visually inspected using Amix 3.9.13 (Bruker, Biospin). 1H NMR spectra were segmented in rectangular bins of fixed 0.04 ppm width and integrated using the Bruker Amix 3.9.13 (Bruker, Biospin) software. For aqueous extracts, the spectral region between 4.92 and 4.68 ppm (residual protic water signal) was discarded and the remaining 232 buckets in the range of 10.00–0.50 ppm were normalized to total area to minimize small differences and subsequently mean-centered. For lipid extracts, the spectral regions between 7.60 and 6.90 (signals of the residual non-deuterated chloroform and its carbon satellites) and 3.60 and 3.00 ppm (due to the possible presence of residual methanol signals in this range) were discarded and the remaining 211 buckets in the range of 10.00–0.30 ppm were normalized to total area to minimize small differences and subsequently meancentered. 2.4. Statistical analysis NMR spectra were analyzed by multivariate statistical procedures. Input variables were generated via binning performed on 1 H zg and 1H cpmgpr1d spectra for lipid and aqueous extracts, respectively. The description of statistical analyses refers to Pareto scaled data. Multivariate statistical analysis and graphics were obtained using Simca-P version 13.2 (Umetrics, Sweden) using PCA and PLS-DA procedures. Principal components analysis (PCA) was performed to examine the intrinsic variation in the data set. Preliminary PCA ordinations of samples were performed for evaluating the contribution of each NMR signal related bin, by means of the respective loading (in the loading plot), to the inter-sample variance characterizing the data set. To maximize the separation between samples, partial leastsquares discriminant analysis (PLS-DA) was applied. The PLS-DA is the regression extension of PCA, which gives the maximum covariance between the measured data (X variable, NMR signal related bin in spectra) and the response variable (Y variable, class membership). The confidence level for membership probability was considered to be 95% (observations at 2.0) and p(corr) (|p(corr)| > 0.5) was performed. By analyzing the B-plot (data not shown), the discriminating variables for each species were assessed. In particular, E. verrucosa showed a higher content of DUFAs and PUFAs (buckets at 2.06, 2.1, 2.82, and 5.38 ppm), C. pagurus exhibited a higher level of buckets at 1.18, 1.22, 0.82 and 0.86 ppm assigned to saturated fatty acids (SFAs) and C. sapidus was characterized mainly by a higher concentration of MUFAs (buckets at 1.3, 1.58, 2.02, 2.26 ppm). A crucial dietary role of the aforementioned FAs in human diet are acknowledged for each lipid class: PUFAs have positive effects in hypercholesterolemia treatment; MUFAs, among all the FAs, have lower tendency to lipo-peroxidation and SFAs are risk factors that predispose to cardiovascular disease (Berry et al., 1991; Williams, 2000). PLS-DA, applied to lipid fractions of crab claw muscle tissues, indicated considerable inter-specific differences in nutritive properties of the studied crustaceans. Representative one-dimensional (1D) 1H NMR spectra of aqueous extracts of the three crab species are shown in Fig. 2. The PCA on aqueous extracts explained more than 82% of the total variance with four components, giving R2 = 0.82 and Q2 = 0.49. In particular, PC1, PC2, PC3 and PC4 explained 40.9, 19.3, 15.6, and 6.8% of the total variance, respectively. In the PC1/PC2 score plot (Fig. 4A) marked among-species differences were observed. C. sapidus clearly separated along the PC1 with respect to the E. verrucosa and C. pagurus, whereas the latter two species differed along the

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ATP

Glycine Homarine

Betaine Trigonelline

Betaine

Tyrosine Phenylalanine

Alanine

Homarine 9.0

8.5

8.0

7.5

7.0

Glutamate

ppm

Taurine Glutamine

C.sapidus

Trigonelline Proline

Methionine Lactate/ Threonine Arginine Isoleu/Leu/Val

E.verrucosa

C.pagurus

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm Fig. 2. Representative 1H NMR spectra in D2O of C. sapidus, E. verrucosa, and C. pagurus aqueous extract.

Table 1 Chemical shift (d) and assignment in the 1H NMR spectrum of lipid extracts. Assignment

d (ppm)

Proton

Cholesterols

0.67, 0.91, 1.00 0.88

C26, C25, C24

0.97 1.18–1.30 1.30–1.38 1.45–1.64 1.64–1.74 1.93–2.05 2.05–2.14 2.21–2.35 2.38 2.75–2.88 3.16 3.30 3.73, 4.30, 4.39, 4.12, 3.92 5.20

CH3 (CH2)n (CH2)n CH2CH2COOH CH2CH2COOH CH2CH@CH CH2CH@CH CH2COOH @CACH2CH2COOH CH@CHCH2CH@CH 8.46 AN(CH3)3 CH2N CH2OP CH2

5.29–5.43

CH@CH

All FAs (SFAs, MUFAs and DUFAs) except n-3 All PUFAs n-3 All FAs All DUFAs and PUFAs All FAs except EPA and DHA EPA (20:5 n-3) All MUFAs, DUFAs and PUFAs All PUFAs n-3 All FAs except DHA DHA (22:6 n-3) All DUFAs and PUFAs Phospatidylethanolamine Phosphatidylcholine Phosphatidylcholine Phosphatidylcholine sn1,3 glycerol of phosphatidylcholine sn2 glycerol phosphatidylcholine Ingrid S. Gribbestadd, Shun Wadae, and Michio Nonakaa All MUFAs, DUFAs and PUFAs

CH3

CH

PC2 axis. The PC1/PC2 loadings plot (Fig. 4B) exhibited a higher level of glycine (3.58 ppm), glutamate (2.02, 2.06 ppm) and alanine (1.5 ppm) for C. sapidus. Higher content of taurine (3.3, 3.34, 3.46 ppm) and homarine (4.38 ppm) were observed for C. pagurus

Table 2 Chemical shift (d) and assignment of the metabolite resonances in the 1H NMR spectrum of aqueous extracts. Metabolites

d (ppm)

Leucine Isoleucine Valine Threonine Lactate Alanine Arginine Methionine Glutamate Glutamine Betaine Taurine Glycine Proline Homarine

0.96 (da, CH3), 0.97 (d, CH3), 1.72 (m, CH), 1.73 (m, CH) 0.94 (t, CH3), 1.01 (d, CH3), 1.98 (m, CH) 1.00 (d, CH3), 1.05 (d, CH3), 2.28 (m, CH), 3.62 (d, CH) 1.34 (d, CH3), 3.64 (m, CH), 4.29 (m, CH) 1.34 (d, CH3), 4.13, (q, CH) 1.50 (d, CH3), 3.80 (m, CH) 1.69 (m, CH2), 1.93 (m, CH2) 2.15 (s, S-CH3), 2.66 (t, CH2), 3.80 (t, CH) 2.05 (m, CH2), 2.35 (m, CH2) 2.13 (m, CH2), 2.46 (m, CH2) 3.29 (s, N(CH3)3), 3.94 (s, CH2) 3.30 (t, CH2NH), 3.45 (t, CH2SO3) 3.59 (s, CH) 4.17 (dd, CH), 2.07 (m, CH2), 2.35 (m, CH2), 3.35 (m, CH2) 4.38 (s, N(CH3)3), 8.00 (dd, C4H ring), 8.06 (d, C6H), 8.57 (dd, C4H), 8.74 (d, C3H) 4.47 (s, N(CH3)3), 8.11 (dd, C4H), 8.86 (dd, C3H and C5H), 9.14 (s, C2H) 6.90 (d, C3,5H ring), 7.19 (d, C2,6H ring) 7.43 (m, C3,5H), 7.39 (m, C4H), 7.33 (m, C2,6H) 8.56 (s, C8H), 8.25 (s, C2H)

Trigonelline Tyrosine Phenylalanine ATP

a Letters in parentheses indicate the peak multiplicities; s, singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplet.

whereas E. verrucosa showed higher concentration of betaine (3.26, 3.94 ppm) and lactate (1.34 ppm) (Fig. 4B). PLS-DA applied to aqueous extracts provided a good descriptive and predictive model (4 PLSs, R2X = 0.82, R2Y = 0.975, Q2 = 0.93, and

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(A)

(C)

(B)

(D)

C. sapidus E. verrucosa C. pagurus

SFAs MUFAs

SFAs MUFAs

PUFAs

Fig. 3. PCA (A) and PLS-DA (C) score plot and relative loading plot (B and D) of bucket table generated by 1H NMR spectra of lipid extracts.

p[CV-ANOVA] = 3.4 * 10 7). As observed preliminarily in the PCA analysis, also in the PLS-DA score plot (Fig. 4C) the blue crab separated from the other two species along the PLS1, whereas the PLS2 discriminated the warty crab from both the blue crab and the edible crab (C. pagurus). In this case the combination of VIP (VIP > 2.0) and p(corr) (|p(corr)| > 0.5) was used to recognize the variables with the highest influence for discriminating the three species. The analyses of loadings plot (Fig. 4D) and B-plot (data not shown) confirmed and validated the data obtained by PCA model with the following differences among species: higher level of alanine, glycine, and glutamate for the blue crab, higher concentration of betaine and lactate in the warty crab, and higher taurine and homarine contents in the edible crab. Interestingly, MVA of NMR data resulted in a better discriminating ability among species for aqueous with respect to lipid fractions. In general, alanine, glycine and glutamate are identified as functionally important amino acids involved in several physiological processes and in some cases useful in the treatment of human pathological states (Gülçin, 2012). In addition, it has been indicated that in different food products alanine and, most importantly, glutamate derivatives promote the ‘‘umami flavor” enhancing artifi-

cially added or naturally present flavors (Ye et al., 2012). Lactate, betaine, taurine and homarine are relevant macromolecules in crustacean physiology and were observed at high levels in E. verrucosa and C. pagurus. Although scant information are available on homarine, the properties of betaine, taurine and lactate are well documented. Specifically, (i) betaine functions as an osmo-protector, participates in methionine cycle, and has a regulative effect on liver activity; (ii) taurine is an intermediate factor in membrane stabilization processes, it stimulates glycolysis and glycogenesis and acts as a powerful anti-oxidant; (iii) lactate is a reliable indicator of mechanical stress and freshness of sea food products; (iv) betaine, taurine and homarine are crucial in the physiology of marine invertebrates, triggering exchange of methyl groups among compensatory osmolytes, in order to protect endogenous biomolecules from sudden and challenging environmental variations (Tikunov et al., 2010; Weimer & Slupsky, 2013; Willmer, Stone, & Johnston, 2009). These results indicate that the adaptive response in C. sapidus may be weaker with respect to both E. verrucosa and C. pagurus. This suggests that the blue crab may result a more tolerant species with respect to stresses due to capture, transport, and distribution across fish markets.

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(C)

(A)

(B)

C. sapidus E. verrucosa C. pagurus

(D)

Lactate

Taurine

Betaine

Homarine

Alanine Glutamate Glycine

Glycine

Glutamate Taurine

Alanine

Betaine Homarine Lactate

Fig. 4. PCA (A) and PLS-DA (C) score plot and relative loading plot (B and D) of bucket table generated by 1H NMR spectra of aqueous spectra.

Probably, the inter-specific dissimilarity among the three brachyuran species, underlined from NMR spectroscopy and MVA, is likely to depend on the susceptibility of these marine invertebrates which are able to change internal biochemical processes according to environmental variations. Indeed, it is worth noting that these species are able to modulate physiological metabolite concentration in order to face the abiotic factors variation in the habitats, the nature of available trophic resources, the genetic diversity, the life stages, and sex. In particular, modulation of amino acidic deposits and nitrogen compounds in crustaceans is triggered by environmental salinity fluctuation producing osmoadaptive processes, while in aquatic organisms a discriminant event during lipid synthesis is the homeoviscous process which regulates membrane fluidity through lipid saturation (Willmer et al., 2009). Finally the description of nutritive components of crab meat, as found in this study, (which is the first comparative work using metabolomic of both lipid and aqueous extract of raw crab claw muscles), offers some interesting indications related to nutritional aspects. Our results contrast with the common idea, among consumers, that crabs and other crustaceans are a source of essen-

tially damaging lipids; conversely, in providing novel and exhaustive information on the nutraceutical value of these foods, they sustain their consumption and commerce. In addition, since C. sapidus is an invasive alien species (IAS) in Mediterranean regions (Nehring, 2011), its consumption could attenuate damaging effect in soft bottom benthic communities of coastal areas in Mediterranean Sea as well as reduce the fishing effort carried on others autochthonous species, included E. verrucosa. 4. Conclusion Crustaceans are becoming the new target of world fisheries to cope with the growth of human populations at a global scale and substitute high trophic level species, such as finfish, depleted by overfishing. Blue crab and edible crab fisheries in North Atlantic are to date well established, yet future scenarios forecast the development of crustacean fisheries also in the Mediterranean Sea. C. sapidus, even though of alien origin, is to date well established and already commercially exploited in the Eastern Mediterranean. The present work reports for the first time the principal metabolomic characteristics of the species in Europe by

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means of 1H NMR spectroscopy and MVA. Interestingly, Blue crab meat showed potentially beneficial contributions to human nutrition due to higher content of PUFAs and lower SFAs when compared to the other two species (E. verrucosa and C. pagurus). Moreover, increased level of taste enhancer such as glutamate and alanine and lower content of compensatory osmolytes (betaine, taurine and homarine) were also found in C. sapidus meat. In particular, the latter point suggests an improved tolerance to manipulation stresses in C. sapidus due to capture, transport, and distribution across fish markets. Even though limited, in terms of the number of specimens analyzed and in the scrutiny of the influence of biotic (e.g., sex) and abiotic (e.g., seasonality) factors on the investigated species, the present study may represent a first, original contribution supporting the adoption of metabolomic analyses and, in general, foodomics approaches for shellfish nutritional characterization and quality assessment. In addition, by presenting novel, advanced information on the chemistry of C. sapidus and E. verrucosa meat, it provides indications on the quality of these two species as seafood products, that may be useful to decision support systems for the implementation of crustacean fisheries in the Mediterranean Sea.

Acknowledgements This research was performed in partial fulfillment of the requirements for a Master’s thesis in Coastal Marine Ecology and Biology by M.Z. G.M. benefited from F.U.R. funding 2013–2014; F.P.F. was supported by the PON (Programma Operativo Nazionale) grant 254/Ric. Potenziamento del ‘‘Centro Ricerche per la Salute dell’Uomo e dell’Ambiente” Code PONa3_00334. Two anonymous reviewers provided helpful comments on an early draft of the manuscript. This paper is dedicated to Sofia Mancinelli, thy eternal summer shall not fade.

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