Juices and Non-Alcoholic Beverages

Chapter 17

Juices and Non-Alcoholic Beverages Miriam Dı´az-Garcı´a, Maria Rosario Castellar and Jose´ Marı´a Obo´n Departamento de Ingenierı´a Quı´mica y Ambiental, Universidad Polite´cnica de Cartagena, Campus Alfonso XIII, Cartagena, Murcia, Spain

Chapter Outline 1. Introduction 2. Juices and Non-Alcoholic Beverages 3. Quality and Authenticity of Fruit Juices 3.1. Polyphenol Profiles 3.2. Organic Acid Profiles 3.3. Sugar Profiles 4. Techniques and Analytical Methods for Designation of Origin

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4.1. High-Performance Liquid Chromatography 450 4.2. Gas Chromatography 451 4.3. Nuclear Magnetic Resonance Spectroscopy 452 4.4. Infrared Spectroscopy 453 5. Conclusions 454 Acknowledgements 455 References 455

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This chapter focuses on the analytical methodology that can be used to evaluate the regulations of protected designation of origin (PDO) and protected geographical indication (PGI) of juices and non-alcoholic beverages. All the member countries of the World Trade Organization are required to follow the fundamental principles of Geographical Indication (GI) law as outlined by the TRIPS agreement. PDO covers agricultural products and foodstuffs which are produced, processed and prepared in a given geographical area using recognized know-how, while PGI covers agricultural products and foodstuffs closely linked to the geographical area. At least one of the stages of production, processing or preparation takes place in the area. Registration of a juice for PGI can be easier to achieve than for PDO [1] (Figure 1). Comprehensive Analytical Chemistry, Vol. 60. http://dx.doi.org/10.1016/B978-0-444-59562-1.00017-7 © 2013 Elsevier B.V. All rights reserved.

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FIGURE 1 PDO and PGI logos.

FIGURE 2 Logotypes of several fruits with PDO/PGI.

The agricultural products needed for the preparation of juices and nonalcoholic beverages are fruits and vegetables, and therefore, this chapter offers a spectrum of analytical proposals able to link a juice composition with the corresponding geographical area of origin of the corresponding fruit or vegetable. Examples are also included. The analysis of healthy components of juices is also a goal because they can give specific quality characteristics or reputation attributable to their geographical origin [2]. Several difficulties are cited in labelling the geographical origin of a fruit or vegetable, such as the changing climatic conditions between seasons and years, outbreak of diseases and irrigation conditions; however, there are a number of important fruits with PDO/PGI (Figure 2). Examples are apples (apples of Bierzo [3]), citrus (clementines of Calabria [4]), oranges (oranges of Ribera of Sicily [5]), pears (pears of Jumilla [6]), cherries (cherries of Jerte [7]), prickly pears (prickly pear of Etna [8]) and many others. On the other hand, actually, as far as we know, there are no juices labelled with GI protection. The market for fruit juices is centred in single flavour types, mainly orange, apple and pineapple, as well as in multifruit juices made with a mixture of flavours [9]. Even with juices made of a single fruit, the juice industry argues that harvest fluctuations and differences in quality and availability of fruits require a blending of juices of different origins to ensure constant consumer quality. As an example offered by the European Fruit Juice Association [10], apple juice concentrate that is used for the production of apple juice originates from 15 EU member states and 13 other countries on different continents. It means that fruit harvesting regions and

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fruit juice industry are not always located together. Juice producers consider it non-viable economically to elaborate fruit juices with the designation of origin because the raw material comes from different parts of the world. Even though the juice industry is aware that consumers are highly interested in the label of geographical origin of foods, GI protection of juices may have a social and economic importance as well. There is also a growing market for added value juices based on the inherent nutritious and health properties of fruits and vegetables. Health claims and quality and authentication analyses are therefore critical to assign a PDO/PGI to juices and for their commercialization.

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For consumer information and protection, it is important to guarantee the authenticity and the quality of fruit or vegetable drinks. The Codex Alimentarius commission was created by the Food and Agriculture Organization of the United Nations and World Health Organization to develop food standards, guidelines and related texts such as codes of practice, with the main purposes of protecting the health of the consumers and ensuring fair practices in the food trade [11] (Figure 3). The Codex General Standard for fruit juices and nectars (CODEX STAN 247-2005), which was adopted by the Codex Alimentarius Commission during its 28th session held from 4–9 July 2005 (Codex Standard), established, in particular, quality factors and labelling requirements for fruit juices and similar products. Fruit juice is the unfermented but fermentable liquid obtained from the edible part of sound, appropriately mature and fresh fruit or of fruit maintained in sound condition by suitable means including postharvest surface treatments applied in accordance with the provisions of the Codex Alimentarius Commission. A single FAO

WHO

Codex Alimentarius Commission

CODEX STAN 247-2005 FIGURE 3 Operating ‘Codex Alimentarius’ diagram.

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juice is obtained from one kind of fruit. A mixed juice is obtained by blending two or more juices or juices and pure´es from different kinds of fruits. Fruit nectar is the unfermented but fermentable product obtained by adding water with or without the addition of sugars, honey and/or syrups, and/or food additive sweeteners or a mixture of those products. Mixed fruit nectar is obtained from two or more different kinds of fruit. Non-alcoholic beverages represent many beverage categories including carbonated soft drinks, sports and isotonic drinks, energy drinks, fortified beverages and bottled waters. In this chapter, we focus mainly on non-alcoholic beverages made from any kind of fruit or vegetable. Any beverage made with fruit or vegetable juices should be subject to testing for authenticity and quality where applicable and where required. The verification of a sample’s authenticity/quality can be made by the comparison of data for the sample, generated using appropriate methods, with that produced for fruit of the same type and from the same region, allowing for natural variations, seasonal changes and variations occurring due to processing. The sampling and analytical methods used should be standard, and the most important are included in the fruit juices and nectars Codex (CODEX STAN 247-2005). The International Federation of Fruit Juice Producers (IFU) [12] represents the fruit juice industry on a global level as a Non-Governmental Organization (NGO). IFU harmonizes standard analysis and practices for juice products and producers. In general, products must present specific sensory attributes related to a region, elaboration procedure or raw materials. In the case of products with quality distinctiveness labels, such as PDO, it is necessary to specify the characteristics and it is important to prove a difference in quality between noncertified and certified products. In many cases, trained and monitored panels check the sensory quality of the product and decide whether it fulfils the minimum requirements (appearance, flavour, texture, etc.) to be sold under this label according to the expected characteristics [13]. Nowadays, PDO in food and beverage products exists. In spite of this, fruit juices are not characterized specifically for this important quality and authenticity label.

3 QUALITY AND AUTHENTICITY OF FRUIT JUICES The Codex general standard for fruit juices and nectars (CODEX STAN 2472005) defines quality criteria as follows: the fruit juices and fruit nectars shall have the characteristic colour, aroma and flavour of juice from the same kind of fruit from which it is made. The fruit shall retain no more water from washing, steaming or other preparatory operations than technologically unavoidable. The term authenticity is defined as the maintenance of the product’s essential physical, chemical, organoleptical and nutritional characteristics of the fruit(s) from which it comes.

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Fruit content represents the main quality parameter of fruit pure´es, fruit beverages and fruit products in general. The specifications and the labelled composition of fruit-based products have to be met in order to maintain product quality and authenticity. From an economic point of view, product quality is also a major issue of competition between producers. In addition, quality and authenticity are of particular importance with respect to consumer expectation [14]. Non-alcoholic beverages such as soft drinks are elaborated mainly with water, fruit juices and food additives that contribute to the final colour and flavour because these are not natural products. Strict regulations about the use of food additives in beverages exist in every country and their use in juices is forbidden. Juices and nectars are characterized for their natural composition. In the food industry, end-products must achieve a compromise between several properties, including sensory, sanitary and technological properties. Among them, sensory and sanitary properties are essential because they influence consumer choice and preference. Nevertheless, managing these properties right from the fabrication stage with the aim of controlling them is not an easy task. It is not always possible to use databases for controlling food product quality [15]. The fruit juice industry is one of the fastest growing sectors of the worldwide beverage industry. Many different types of fruits are now being processed into juice, but oranges still provide most of the fruit juices and other fruit products sold around the world [16]. Consumers expect manufacturers and retailers to provide wholesome and authentic fruit juices. These factors have underlined the need for reliable techniques that can authenticate the purity of fruit juices. Fruit juices command premium prices and therefore represent favoured targets for adulterations, for instance, blending juices of high-priced fruits with those of cheaper fruits. Products obtained do not show correspondence between their composition and that of the specified fruit in the label. For this reason, numerous attempts at finding suitable methods for authenticity control and determination of the fruit content in fruit-based products have been made. The major analytical problem is the complexity of the products and the substantial variance of the fruit-specific components. Analytical techniques to face this challenge are at least as manifold as are the ways of adulteration, ranging from classical determination of chemical parameters to highly sophisticated instruments and techniques [14]. Analysis of a single natural juice component would be inadequate to provide enough information about the juice; therefore, multiple component chemical analyses are required to evaluate reliably the differences between adulterated and pure juices. This approach, however, is both time consuming and expensive. For determining the authenticity of juices and detecting adulteration, a combination of chemical analyses of juice components, such as polyphenols, organic acids, sugars, amino acids and other inorganic components, is done [16].

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The Codex general standard for fruit juices and nectars (CODEX STAN 247-2005) indicates that fruit juices and nectars should be subject to testing for authenticity, composition and quality where applicable and where required. The verification of a sample’s authenticity/quality can be assessed by the comparison of data for the sample, generated using appropriate methods included in the standard, with that produced for fruit of the same type and from the same region, allowing for natural variations, seasonal changes and variations occurring due to processing. CODEX STAN 247-2005 proposes many different analytical methods for the assessment of food authenticity and detection of adulteration. Table 1 shows these methods. We want to focus on polyphenols, organic acids and sugar profiles as important fruit juice components to assess its quality and authenticity.

3.1 Polyphenol Profiles There is epidemiological evidence linking a diet rich in fruits and vegetables with health benefits. Attention is focused on the prevention of degenerative diseases, cardiovascular diseases and cancer. These benefits have been associated with some groups of polyphenol compounds. The main antioxidants of human diet are constituted by polyphenols, and the health effects of polyphenols depend on the amount consumed and their bioavailability [17]. Several thousands of plant polyphenols are known, including a wide variety of molecules that contain at least one aromatic ring with one or more hydroxyl groups in addition to other constituents [18]. The International Fruit Juice Association (IFU) does not have a total polyphenol method to analyse profiles and quantify polyphenols [12]. Thus, it is crucial to have an easy and powerful analytical methodology to measure the polyphenol content of commercial fruit juices because the isolation and quantification of polyphenols in fruit juices and beverages, in general, are difficult due to the chemical complexity. Several analytical methods have been developed including high-performance liquid chromatography (HPLC) [19], gas chromatography (GC), high-speed counter-current chromatography [20] and capillary electrophoresis (CE), followed by ultraviolet (UV), electrochemical (EC), fluorescence (F) and mass spectrometric (MS) detection [21]. HPLC is the most widely used method for the analysis of dietary polyphenols. The structural information and confirmation of the identity of fruit juice analytes are obtained in combination with MS [22], for example, detection by HPLC–PDA–MS fingerprinting to assess the authenticity of pomegranate juices [23]. An important group of polyphenols present in red fruit juices is anthocyanins. Anthocyanin profiles are used as fingerprinting for red fruit juice authentication. The quality of a red fruit juice is also related to its anthocyanin content. The specific method to determine anthocyanins is the IFU Method

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TABLE 1 Several Methods of Analysis and Sampling (CODEX STAN 247-2005) Provision

Method

Principle

Type

Anthocyanins (quality criteria and authenticity)

IFU Method No. 71 (1998)

High-performance liquid chromatography (HPLC)

I

Beet sugar in the juices (quality criteria and authenticity)

AOAC 995.17

Deuterium nuclear magnetic resonance (deuterium NMR)

II

C13/C12 ratio of ethanol derived from fruit juices (quality criteria and authenticity)

JAOAC 79, No. 1 (1996), 62–72

Stable isotope mass spectrometry

II

Essential oils (Scott titration) (quality criteria and authenticity)

AOAC 968.20 IFU Method No. 45

(Scott) distillation, titration

I

Fermentability (quality criteria and authenticity)

IFU Method No. 18 (1974)

Microbiological method

I

Free amino acids (quality criteria and authenticity)

EN 12742 (1999) IFU Method No. 57 (1989)

Liquid chromatography

II

High fructose corn syrup and hydrolized inulin syrup in apple juice (permitted ingredients)

JAOAC 84, 486 (2001)

Capillary gas chromatography

IV

L-Malic/total

malic acid ratio in apple juice (quality criteria and authenticity)

AOAC 993.05

Enzymatic determination and highperformance liquid chromatography (HPLC)

II

Proline by photometry— non-specific determination (quality criteria and authenticity)

EN 1141 (1994) IFU Method No. 49 (1983)

Photometry

I

Sodium, potassium, calcium and magnesium in fruit juices (quality criteria and authenticity)

EN 1134 (1994) IFU Method No. 33 (1984)

Atomic absorption spectroscopy

II

No. 71 (1998) by HPLC with UV–visible detection. As an example, Figure 4 shows a PDA chromatogram of blueberry juice (Vaccinium myrtillus L. wild). Although anthocyanins are also detected in the UV region, usually they are analysed at 520 nm. If we obtain the 520-nm chromatogram from the PDA as

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Blueberry PDA 14

13

nm

500.00

400.00

300.00

2.00

4.00

6.00

8.00

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FIGURE 4 PDA chromatogram of blueberry juice (Vaccinium myrtillus L. wild).

shown in Figure 4, it is possible to observe an efficient separation of 14 anthocyanins where peak assignments are as follows: (1) delphinidin 3-galactoside, (2) delphinidin 3-glucoside, (3) cyanidin 3-galactoside, (4) delphinidin 3-arabinoside, (5) cyanidin 3-glucoside, (6) petunidin 3-galactoside, (7) cyanidin 3-arabinoside, (8) petunidin 3-glucoside, (9) peonidin 3-galactoside, (10) petunidin 3-arabinoside, (11) peonidin 3-glucoside, (12) malvidin 3-galactoside, (13) malvidin 3-glucoside and (14) malvidin 3-arabinoside [24]. The amount and diversity of polyphenols in vegetable tissues cause the difficulty in obtaining pure profiles with no peak overlapping in HPLC chromatograms. The different detection systems are important to analyse and corroborate the identification of every compound. IFU Method No. 71 improved by using both UV–visible and fluorescence detection can determine a higher number of polyphenols different from anthocyanins. Chromatograms using fluorescence detection are more specific than absorbance chromatogram for some fruit components. As an example, Figure 5 shows the identification in blueberry juice of (1) gallic acid, (2) 3,4 dihydroxybenzoic acid, (3) (þ)catechin, (4) ()-epicatechin, (5) vanillic acid, (6) syringic acid and (7) resveratrol-glucoside. Fluorescence detects five additional polyphenols [24]. To simplify peak identification and quantification analysis, some authors hydrolyze juice polyphenols before HPLC analysis. For example, Mattila et al. [25] made an alkaline hydrolysis in their studies to determine polyphenol content in European blackcurrant juices. In these cases, authentication of fruit juice is more difficult because the determined compounds are not the original components of the analysed juice. In addition, some specific methods exist for the analysis of juice profiles of thedifferent polyphenol types: anthocyanins, procyanidins, flavanones, flavonols, flavan-3-O-ols, flavones and phenolic acids [26]. However, special emphasis is focused on a general fast HPLC method for all polyphenol analysis [27–29], valid for fruit juice authentication.

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0.20

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2

Abs 260 nm

Anthocyanins Other compound

AU

0.15

0.10

0.05

0.00

1

80.00

4

2

Fluorescence:

5

Fluorescence

60.00

lexc = 290 nm ; lemi = 350 nm

6

3 40.00

7 20.00 0.00

2.00

4.00

6.00

8.00

10.00 12.00

14.00 16.00

18.00 20.00

22.00 24.00

26.00 28.00 30.00

min

FIGURE 5 HPLC chromatograms of blueberry juice obtained using both UV–visible and fluorescence detection.

In a similar way, capillary electrophoresis with electrochemical detection has been reported to determine the polyphenol compounds in apple juices and ciders. Phloridzin, ()-epicatechin, chlorogenic acid and myricetin could be separated within 20 min in a 75-cm-length capillary at a separation voltage of 18 kV in a 50-mmol/l borate buffer (pH 8.7), and a 300-mmdiameter carbon disk electrode generated a good response at þ0.90 V for all analytes [30].

3.2

Organic Acid Profiles

The determination of low molecular weight organic acids in fruit juices is important because of their influence on organoleptic properties such as flavour, colour and aroma. Their presence is also important for the stability and microbiological control of these beverages [31]. Organic acid levels are indicators of freshness and product quality. Besides, organic acids are added to some food and beverage systems for enhancing flavour, as acidulants, to control the pH of a commercial product and to be used as antimicrobial agents [32]. The principal acids used to enhance beverage flavours are citric, tartaric, fumaric and phosphoric acids. Citric acid is the most widely used, while malic

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and tartaric acids are important natural compounds of fruits that are used along with fumaric acid in fruit-flavoured drinks [33]. The analyses of these acids allow checking the process of maturation of fruit, to control the evolution of the acidity, and are also important in the testing of authenticity of fruit juices, where organic acid profiles are monitored to determine their purity. These analyses have been especially developed for fruit juice adulteration detection, where organic acid fingerprint is a useful instrument to helpevaluation by comparing the known juice profile to that of the suspected adulterated juice. Tartaric, malic, citric acids and others are naturally found in fruits. Therefore, they can be analysed in fruit juices for authenticity control. Tartaric acid is usually considered an indicator of grape juice for its own authenticity or if it has been added to a more expensive juice. Similarly, malic acid is a major component of the organic acid content of apple juice; excess malic and/or quinic acid can be used as an indicator of apple juice addition to other fruit juices as adulterant. Quinic/citric, quinic/malic and citric/malic ratios are important in cranberry juice authenticity determination. The citric/isocitric ratio is especially important in orange juice, where a ratio of >130 suggests dilution corrected by the addition of citric acid. Isocitrate exists in natural orange juices in small but constant amounts. It is used as an adulteration indicator. The confirmation of tartaric and quinic acids in pomegranate juice is used in its authenticity testing [34]. Analytical methods used routinely for organic acids are based on liquid chromatography reverse phase or ion exchange coupled to UV detection (AOAC official method 986.13 2008) [35]. This chromatographic technique allows simultaneous analysis of most of the organic acids. It is used either for direct determination or for the analysis of derivatized samples. Different detection systems are employed: refractive index (RI), UV spectrophotometric, conductimetric and electrochemical detection [36]. More recently, LC–MS with stable isotope dilution has been applied to the analysis of some organic acids in Vaccinium berries [37]. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) with a stable isotope dilution is also employed for organic acid analysis in fruit juice authentication [34].

3.3 Sugar Profiles As organic acids, sugars present in fruit juices are also involved in very important characteristics, such as taste, flavour, maturity and quality, and are indicative of storage conditions. The determination of sugars in fruit juices also may serve as indicators of the authenticity or adulteration of these foods. Frequently, fruit juices are adulterated by addition of commercial sweeteners, which represents an economic and regulatory problem. The most common forms of adulteration include simple dilution and blending of inexpensive and synthetically produced juices into the more expensive ones. The source of sweetener for adulteration can be other juices or sugar derived

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from fruits or vegetables. Therefore, qualitative and quantitative knowledge of sugar distribution in fruit juices, the minor sugars, the less frequent enantiomers of sugar and the ratio of major sugars or sugar fingerprinting are used for origin and authenticity determination in commercial fruit juice products. Sugars that could be added are sucrose, high fructose corn syrup, cane medium invert syrup and beet medium invert syrup. The development of products resulting from the transformation of sucrose (in cane or sugar beet) and starch (in maize) changes the nature of the problem involved in detecting the adulteration of fruit juices by sugar addition. They are cheap sources of sugars from which it is possible to prepare (by acid or enzymatic hydrolysis) mixtures of sucrose:glucose:fructose which are similar to those usually found in juices. However, secondary products (oligosaccharides) are formed by a process known as ‘reversion’ during the preparation of these derivatives, whether in an acid medium or in the presence of enzymes. Thus, the presence and nature of these oligosaccharides do not depend on the botanical origin of sugars. The profile of these oligosaccharides is conditioned by the preparation method (either enzymatic or by heating in acid medium), by the hydrolysis time and by the concentration and nature of the sugars present in the reaction medium. By studying the profile of oligosaccharides present in the juice, a possible adulteration by addition of modified sugars can be detected [38]. So, the availability of analytical methods to analyse sugar fingerprinting in fruit juices and other non-alcoholic beverages is essential for authenticity determination. For this, several methods have been developed, principally by chromatographic techniques, HPLC and GC. Other related techniques such as high-performance thin layer chromatography with automated multiple development and CE have also been performed [38]. Sugars are difficult to separate by conventional reverse-phase liquid chromatography, and they lack suitable chromophores or fluorescent groups, which limit both the compound separation and the UV–vis or fluorescence detection. HPLC methods with LC-NH2 columns and refractive index (RI) detection have been the most described technique for sugar analysis for several years. Sugar retention decreases as the proportion of water/acetonitrile in the mobile phase is increased. Under these conditions, sugars generally will be eluted in order of increasing molecular weight. RI detection has a limited sensitivity; thus, other detection systems such as evaporative light-scattering detector, electrochemical and infrared detection have also been employed. The study of oligosaccharide fingerprinting employing high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEPAD) provided an important method for authentication and to detect the undeclared addition of a variety of inexpensive sweeteners to fruit juices [39]. HPAE-PAD allows direct quantification of non-derivatized carbohydrates at low picomole levels with minimal sample preparation and clean-up. This has allowed the detection of carbohydrates in a variety of complex matrices, for instance, foods, beverages and diary products [40]. Therefore, the AOAC

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Official Method No. 995.13 for carbohydrate determination is an anionexchange chromatographic method with pulsed amperometric detection (HPAEC-PAD). This method is useful for both fruit juice authentication and adulteration detection. It is rapid and easy to use and has detection limits that may be of the order of 5% in juices which are free of endogenous oligosaccharides. However, in some cases, the results may need confirmation by the stable isotope method. It is essential to have previously analysed authentic industrial juices, to have a collection of adulterants and to perform the analyses under the same conditions, in order to ensure the reliability of the results [38]. Sugars principally quantified are glucose, fructose and sucrose and their ratios. These values can be affected by variety, maturation stage and geographical origin of the fruit used for juice production. Differences in sugar ratios can be indicative of adulteration by addition of exogenous sugars or blending with cheaper fruit juice. Grape juice has high a concentration of glucose and fructose and lower concentration of sucrose. The sugar predominant in apple juice is fructose. In orange and pineapple juices, sucrose is the major sugar. When sugars are added to a juice, the sugar profile changes and can be evidence of adulteration. Sorbitol is a sugar alcohol detected in the fruit juice sugar profiles and present at low concentration in some fruits such as apple, peach and pears. It is used as sweetener and added to fruit juice as adulterant. Its detection in fruit juices is evidence of possible adulteration [41].

4 TECHNIQUES AND ANALYTICAL METHODS FOR DESIGNATION OF ORIGIN 4.1 High-Performance Liquid Chromatography Several foods from different geographical origins have been correctly classified on the basis of the chromatography profiles of some of their compounds. These are usually obtained by HPLC in combination with a UV–vis and/or fluorescence detector [42]. HPLC is widely used for fruit juice characterization and authentication. Many methods are developed without sample derivatization [43]. As an example of fruit juice, pure Valencia orange (Citrus sinensis L.) juice from Spain and Belize was studied by a liquid chromatographic method. Quantification of the major carotenoid pigment showed clear differentiation between them and was useful to establish different geographical origins. In this way, this method, using a reverse-phase C-18 column, can be a valuable tool for orange juice quality control. In addition, the method also can determine polyphenolic profiles. These are not practical for origin differentiation but permit authentication of citrus juices. Results show a difference between both origin samples based on carotenoid analysis; the pure Valencia orange juice from Spain contains a higher total carotenoid content than Belize oranges [44].

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Profiles of different polyphenol groups are used to find the geographical origin of some fruits. Hydroxycinnamic acids are one of these polyphenol groups. A study of the distribution of hydroxycinnamic acids as a criterion to evaluate variety and geographical origin of Italian orange (C. sinensis L.) juices has been performed. In this study, hydroxycinnamic acids, ferulic, p-coumaric, sinapic and caffeic were determined in 113 orange juices. Their concentration was evaluated to develop a data bank of Italian juices and use it to check variety and geographical origin of commercial products. Results show that p-coumaric acid content was the determining factor in differentiating the juices [45]. Other authors studied anthocyanins in Turkish and Finnish bilberries (V. myrtillus L.). The anthocyanin profiles were analysed by RP-HPLC-DAD. Results from both fruit juices showed different sugar moiety proportions according to the origin. The significant differences in the proportions of these molecules were used for a preliminary model using logistic regression. This model can be used for geographic origin determination. The logistic regression model based on glucose proportion classified 96.7% of samples correctly into their geographical origin. Therefore, the analysis of anthocyanin glucoside moieties provides a novel authentication tool for confirming the geographical origin [46]. Anthocyanidins are the anthocyanin aglycones, and their profiles too can differentiate fruit origin. The effects of latitude-related factors and geographical origin on anthocyanidin concentrations in V. myrtillus L. fruits were studied. The HPLC analysis showed that anthocyanidin concentrations varied significantly with latitude and geographical origin in northern Europe, with higher values from northern latitudes or from a more northerly origin of parent plants. The results show that anthocyanidin concentrations in bilberries are under strong genetic control but are also influenced by climatic factors. Furthermore, the proportions of specific anthocyanidins differed between latitudes and plants with different parental origins [47]. Geographical origins of apricot fruits from several common cultivars and wild type, grown in different regions of Turkey, were studied by an HPLC– UV method able to determine the vitamins A, C, E and b-carotene. In addition, selenium levels were analysed by a fluorometric method. The results show that, in general, the vitamins, b-carotene and selenium levels were found to be significantly different, both between varieties and between different regions for the same variety. Cultivars in a high altitude region had significantly higher vitamin C content than the same cultivars grown in other regions [48].

4.2

Gas Chromatography

GC is another technique used in food analysis, mainly in volatile and semivolatile composition studies, aromas and pesticides [42]. Determination of

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the fatty acid composition and its corresponding concentrations by GC allows the geographical discrimination of fat samples such as milk [49], olive oil [50] and cocoa [51]. In the case of fruit juices, orange juices have been studied via GC analysis. This work was examined from two different approaches: steam distillation– solvent extraction–GC–MS and solid-phase microextraction–GC in orange juices (C. sinensis L.) with different geographical origins. The two sample preparation techniques were compared and most target compounds exhibited constant enantiomeric ratios in all juices when either of the two approaches was used. Exceptions were found for terpinen-4-ol and beta-citronellol, whose enantiomeric purity ratios varied significantly according to the geographical origin of the sample [52].

4.3 Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance (NMR) is based upon the measurement of absorption of radiofrequency radiation by atomic nuclei with non-zero spins in a strong magnetic field [53]. The most commonly studied nuclei are 1H and 13C. 1H NMR spectroscopy is a well-known rapid screening method and has been demonstrated to be efficient in beverage authentication [42]. Investigations of the ratios of stable isotopes and the contents of unstable isotopes (radioisotopes), especially heavy elements, have been used primarily in geological sciences for age determination [54] and to determine and verify the geographical origin of food products [55]. For instance, isotope ratio measurements have been studied for origin designation for many kinds of foods such as meat [56], Chinese cabbages [57], rice [58] and Mediterranean olive oils [59,60]. Regarding fruits, isotope ratio studies discriminate orange juice from grapefruit juice and blends in a context of fraud prevention as an alternative to chromatographic methods. The differences in the 1H NMR spectra of similar samples are useful for authentication of foods [61]. Also, fruit pure´e adulteration can be detected by NMR [62]. As an example, Slovenian apples were characterized by the combination of multi-element analysis, several isotopic ratios (13C/12C, 15N/14N, 18O/16O, 2 H/1H) and selected chemical and physical parameters (fruit mass, antioxidant activity, content of ascorbic acid and total phenols). These analyses were used to differentiate the varieties of Slovenian apples, the geographical location of their growth and agricultural practice (organic or conventional). The stable isotope parameters in sugar, pulp, protein and water were shown to be the most significant variables. Botanical origin (cultivar) was found to have a major influence on the d13C and d15N values of proteins and the d18O and dD values of water. Geographical regions were well separated based on the d18O and dD values in water and the concentrations of Rb and S in fruit juice. The most significant variables to distinguish between organically and

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conventionally cultivated fruits were found to be dant activity [63].

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N/14N ratio and antioxi-

Infrared Spectroscopy

Infrared spectroscopy (IR) is the measurement of the wavelength and intensity of the absorption of infrared light by a sample [64]; in fact, IR measures the vibrations of molecules. IR spectroscopy is a non-invasive and nondestructive technique [65], rapid and can be easily applied in fundamental research, in control laboratories, and online in the factory to analyse food products. Several examples of geographical classification by IR analysis are rice [66], olive oil [67] and fruit such as grapes [68]. Regarding fruit juice authentication, infrared technology has gained acceptance as a rapid and reliable technique. It can be used to evaluate the overall sample composition, providing a fingerprint that is characteristic of the product, and it could be used to determine addition of foreign materials, to differentiate juice commodities or even to evaluate geographical origin of a juice [69]. For instance, there are several studies on pomegranate juice concentrate with Fourier transform infrared spectroscopy and chemometric techniques to detect adulteration with grape juice concentrate. The main differences between both infrared spectra occurred in the 1780–1685 cm1 region, and the analysis was used to differentiate pure juice samples and to classify adulterated samples [70]. Atomic spectroscopy can be used to analyse the vaporized atoms of metals and non-metals in a variety of samples. In atomic absorption spectroscopy, the absorption of light is used to measure the concentration of gas-phase atoms [71]. Chemometric analyses have enabled the determination of the geographical origin of various food products; for instance, selenium content of beef was related to a geographical region [72]. Atomic spectroscopy was also useful for PDO authentication of honey [73], olive oil [74] and potato [75]. As an example, in fruit juices, samples of clementine variety ‘Comune’ with PGI brand ‘Clementine of Calabria’ were studied and stepwise linear discriminate analysis, soft independent modelling of class analogy and partial least-squares discriminant analysis were used to build chemometric models able to differentiate PGI clementine from others of different origins [76]. Sensor technology, sometimes referred to as ‘electronic nose technology’, is based on the detection by an array of semi-selective gas sensors of the volatile compounds present in the headspace of a food sample [77]. This headspace is injected into the detection system of the electronic nose which consists of a sensor set. The advantages of electronic nose technology include a relatively small amount of sample preparation, a simple procedure, and a fast and cheap analysis [56].

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A disadvantage of this technique is that the identification of the chemical compounds detected is not possible and that the detection limit is high compared to that of other methods such as GC–MS. Nevertheless, the electronic nose has been successfully applied for differentiation on the basis of geographical origin in olive oils [78,79], cheese [80] and saffron samples from different countries (Spain, Iran, Morocco and Greece) [81]. Regarding juices, sensor arrays were used for origin authentication of 49 pure Valencia orange (C. sinensis L.) juices from 5 different origins, representative of the main culture areas of citrus according to their volatile organic fractions. Sensor arrays performed a good discrimination of the whole juices in classing them according to the origin of Valencia oranges used. This technique coupled with other ones such as HPLC and GC may be a new tool for the investigation of adulteration detection due to the authentication of the origin of raw materials employed in the orange juice processes [82]. The polymerase chain reaction (PCR) is a biological technique, allowing the detection of very low amounts of nucleic acid probes and the determination of their sequence via the amplification of DNA or RNA molecules [53]. This technique is used extensively to identify the species of origin in foods. PCR has the advantage of high sensitivity and rapid performance with high sample numbers being automatically processed [83]. It has been used for the geographical discrimination of grapes [84], olive oil [85], salmon [86] and can be used to detect juice ingredients from fruits [87]. DNA-based analysis for the authentication of fruit juices has been used to detect grapefruit or mandarin juices in orange juice [88].

5 CONCLUSIONS There are a few fruit juices and non-alcoholic beverages with PDO or PGI designation. This is a consequence of the diverse origin of raw material used by industry for the manufacture of these products. Their analysis is necessary for authorities interested in sanitary control and in the correct labelling of food products and for consumers interested in knowing their composition, properties and origin. With regard to fruit juices and non-alcoholic beverages, several analytical methodologies have been developed for authentication and some have been applied to PDO or PGI product analysis. The profiles or fingerprinting of different compounds obtained by HPLC, GC and IR spectroscopy and the isotope ratio, sensor technology and PCR methodologies can contribute to PDO or PGI control and designation. However, it is difficult to use a unique technique for this purpose. Some analytical procedures may determine quality or authentication of fruit juice, whereas to establish the geographical origin, it is necessary to combine different methodologies and work with chemometric analysis, including linear discriminate analysis, principal components analysis and others.

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ACKNOWLEDGEMENTS The present work has been supported by ‘Fundacio´n Se´neca’ (18643/BPS/12 and 08702-PI-08). The authors thank J. Garcı´a Carrio´n, S.A. (Jumilla, Spain) for their contributions.

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