Determination of the Geographical Origin of All Commercial Hake

Jan 15, 2017 - The specific results enabled their clear classification according to the FAO (Food and Agriculture Organization of the United Nations) ...
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Determination of the Geographical Origin of All Commercial Hake Species by Stable Isotope Ratio (SIR) Analysis Mónica Carrera, and José M. Gallardo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04972 • Publication Date (Web): 15 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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Journal of Agricultural and Food Chemistry

Determination of the Geographical Origin of All Commercial Hake Species by Stable Isotope Ratio (SIR) Analysis Mónica Carrera1*, José M. Gallardo1

1

Marine Research Institute (IIM), Spanish National Research Council (CSIC), Vigo,

Pontevedra, Spain

AUTHOR E-MAIL ADDRESS: [email protected]

*

CORRESPONDING AUTHOR: Dr. Mónica Carrera

Marine Research Institute (IIM), Spanish National Research Council (CSIC), Eduardo Cabello 6, 36208, Vigo, Pontevedra, Spain. Phone: +34 986231930. Fax: +34 986292762. E-mail: [email protected]

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ABSTRACT

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The determination of the geographical origin of food products is relevant to comply

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with the legal regulations of traceability, avoid food fraud and guarantee the food quality and

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safety to the consumers. For these reasons, Stable Isotope Ratio (SIR) analysis using an

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isotope ratio mass spectrometry (IRMS) instrument is one of the most useful technique for

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evaluating food traceability and authenticity. The present study was aimed to determine for

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the first time, the geographical origin for all commercial fish species belonging to the

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Merlucciidae family using SIR analysis of carbon (δ13C) and nitrogen (δ15N). The specific

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results enabled their clear classification according to the FAO (Food and Agriculture

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Organization of the United Nations) fishing areas, latitude and geographical origin in the

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following six different clusters: European, North African, South African, North American,

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South American and Australian hake species.

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KEYWORDS: Fish; Merlucciidae; Geographical Origin; Stable Isotope Ratio; SIR; δ13C;

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δ15N; Traceability

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Journal of Agricultural and Food Chemistry

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INTRODUCTION

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The consumption of fish is increasing, reflecting strong evidence of the positive

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benefits of these organisms in human health. These benefits primarily reflect the high content

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of polyunsaturated ω-3 fatty acids, which aids in the prevention and treatment of

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cardiovascular, neurological and inflammatory diseases.1 In developing countries, the average

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annual per capita consumption of fish is 9.0-15.1 kg (FAO, 2010).2

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Attributable to this high demand, the fishery market is showing a similar dramatic

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growth. Currently, fish are produced in one country, processed in a second country and

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consumed in a third country. The process of market globalization has created substantial

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opportunities, but these opportunities are associated with inherent risks. A common fraudulent

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practice is species substitution, which could be unintentional, but is more frequently practiced

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for tax evasion, illegally laundering caught fish or selling low-priced fish species as a

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substitute for more valuable high-priced species. In addition, potential human health risks

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include harmful or aggravated symptoms in humans with sensitive allergies. Indeed, fish

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species are one of the foods with a major prevalence of food allergy.3

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In the European Union, labeling regulations are managed by the EU Regulation

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1379/2013 on the common organization of the markets in fishery products.4 This regulation

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advises that fish should be correctly labeled indicating (i) the commercial designation of the

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species, (ii) the production method (caught or farmed) and (iii) the geographical origin (the

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catch area). For this purpose, the Member States publish a list of commercial designations

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accepted in territories indicating the scientific name for each species and the name in the

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language or languages of the Member State. The indication of the geographical origin is

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normalized in the Commission Implementing Regulation (EU) No. 1420/2013.5 For this

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reason, the geographical origin of marine fishery products is guaranteed based on the labeling

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of the number of major fishing catch areas registered by the Food and Agriculture

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Organization (FAO). Internationally, the FAO established a total of 27 major fishing areas

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distributed worldwide. These requirements have been implemented in each of the European

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States, such as Spain, where several regulations have been promulgated to assure the correct

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labeling and identification of seafood products (Royal Decree 121/2004; Royal Decree

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1702/2004).6,7

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To comply with these regulations, accurate, sensitive and fast detection methods are

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essential to confirm the geographical origin and authentication of the fishery products. Many

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different instruments and molecular methods have been applied for fish authentication,8

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including chromatography by HPLC, infrared spectroscopy, NMR spectroscopy, DNA-based

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methods,9 and recently proteomic methods.10,11. However, for the precise determination of

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geographical origin of food products, stable isotope ratio (SIR) analysis is the technique more

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accuracy and preferably used because is only influenced by the relative distribution of

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isotopes in the nature and also provide relevant information about the history of feeding

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relationships through the trophic chain.8,12

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Thus, over the last years, SIR analysis has become the most used technique to assess

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the geographical origin of foods.8,12 The measurement of the isotope ratio has been revealed

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as unique and relevant fingerprints. Indeed, SIR has also been useful for studying the

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geographical origin of olive oil, wines, honey, fruit juices, meat and milk,8,13-15 but was

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scarcely used in fish,16,17 and shellfish.12 In the case of seafood, most of these works applied

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SIR analysis for the discernment of farmed and wild fish species, as salmon fish species.18,19

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Concerning the identification of species, the SIR of carbon and nitrogen was used to

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discriminate Atlantic cod specimens.20 This analysis takes advantage of natural variations in

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the ratios of

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nitrogen propagate from prey to predator through the trophic chain,

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C to

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C (δ13C values) and

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N to

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N (δ15N values). Because carbon and 13

C/12C and

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N/14N

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Journal of Agricultural and Food Chemistry

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indicate a history of feeding relationships, as two of the most informative parameters in the

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diets of animals, and therefore, these parameters can be used as a proxy for geographical

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origin determination.

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Thus, the goal of the present work was to discern the geographical origin using SIR of

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carbon (δ13C values) and nitrogen (δ15N values), applied for the first time to all commercial

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fish species belonging to the Merlucciidae family.

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MATERIALS AND METHODS

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Hake species

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A total of 60 specimens belonging to the 13 main commercial hake species distributed

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worldwide were employed in the present study (Table 1). Thus, all the main commercial fish

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species belonging to the Merlucciidae family were sampled in this work. The specimens were

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purchased to FREIREMAR S.A. fishery company (Las Palmas, Spain), which has fishing

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grounds in all the different FAO fishing areas worldwide (Figure 1 and 2). The irrelevant

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stock species Merluccius angustimanus and M. albidus were not included (no or minor

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commercial). Whole specimens were frozen at -30 ºC on board by FREIREMAR S.A. with

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special care in maintaining the morphological characteristics; and then the fish were shipped

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by plane to the laboratory (6-24 h). The weight of every specimen studied ranged from 3-6 kg.

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The specimens were subjected to taxonomical study according to the anatomical and

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morphological features assessed by an expert marine biologist and by a genetic identification

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in the Food Biochemistry Laboratory at the Marine Research Institute (Vigo, Pontevedra,

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Spain) using the fishID Kit (Bionostra SL., Madrid, Spain). Two replicate per sample were

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analyzed.

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No ethics approval was necessary because the specimens were obtained from conventional extractive fishing activities dedicated for human consumption.

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Stable Isotope-Ratio analysis

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The muscle tissue of each sample was dissected and stored in a frozen state in a deep

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freezer (-80 ºC). The samples were dried at 60 ºC during 72 h, pulverized to a fine powder

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with a ball mill grinder and kept in a glass desiccation vial. For the SIR analysis of

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and 15N/14N, 0.2 g of dried, ground tissue was defatted according to Folch et al. (1957).21A

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C/12C

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Journal of Agricultural and Food Chemistry

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total of 10 mL of CHCl3/CH3OH (2:1, v/v) was added, and after agitation for 10 min, the

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sample was incubated for 12 h at 4 ºC. The samples were subsequently centrifuged (100 x g,

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10 min), and the supernatants were discarded. The samples were subjected to three

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extractions, and after that were dried for 12 h. The dried residue was cleared three times with

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10 mL of MilliQ water and was centrifuged for 10 min at 150 x g. The pellet was dried for 12

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h at 50 ºC and pulverized again. A total of 2 mg of the homogeneous dried material was

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analyzed using an Isotope Ratio Mass Spectrometer (IRMS) (Thermo Scientific, MAT 253,

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Bremen, Germany). The stable isotope ratio values were expressed with delta (δ) notation in

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parts per thousands (‰) relative to the international reference materials. Vienna PeeDee

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Belemnite (V-PDB) (abundance ratio: 1.1237 x 10-2) was used as a reference for carbon, and

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atmospheric nitrogen (N2) (abundance ratio: 3.677 x 10-3) was used as a reference material for

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nitrogen. Stable isotope ratios δ13C and δ15N were expressed according to the following

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equations:

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δ13C (‰)=[(13C/12C)sample/(13C/12C)standard – 1] x 1000

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δ15N (‰)=[(15N/14N)sample/(15N/14N)standard – 1] x 1000

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The IAEA-600, reference standard (caffeine reference standard) approved by the 13

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International Atomic Energy Agency (IAEA), was used for

C analysis. The results of the

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13

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sample relative to the IAEA-600 reference standard. Thus, a δ13C value of –20‰ indicates

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that the content of 13C in the analyzed sample is 20‰ lower than in the reference standard.

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The analysis of variance (standard deviation; s.d.) was