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Authentication and Traceability of Fruits and Vegetables
Chapter 18
Authentication and Traceability of Fruits and Vegetables Yaeko Suzuki* and Rumiko Nakashita{ *
Food Analysis Laboratory, Analytical Science Division, National Food Research Institute, National Agriculture and Food Research Organization (NARO), Kannondai, Tsukuba, Ibaraki, Japan { Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan
Chapter Outline 1. The Geographical Indication System of Fruits and Vegetables Around the World 461 2. Discrimination of Organic and Conventional Fruits and Vegetables 463
3. Tracing the Geographical Origin of Fruits 4. Tracing Geographical Origin of Vegetables 5. Future Perspectives in PDO Authentication of Fruits and Vegetables References
467 470
473 475
1 THE GEOGRAPHICAL INDICATION SYSTEM OF FRUITS AND VEGETABLES AROUND THE WORLD Fruit and vegetables play an important role in a healthy, balanced diet. They contain vitamins, minerals, fibre, sugar, as well as other minor nutrients. The types and amounts of available fruits and vegetables have been expanding globally, and consumers can now obtain products from all over the world. However, in recent years, consumers have experienced numerous negative incidents such as unsafe levels of residual pesticide in exported fruits and vegetables, which has prompted greater awareness of geographical origin and chemical levels in food. Such concerned consumers prefer to buy fruits and vegetables directly from farmers even if the products are more expensive than exported equivalents. The increased demand for reliable food in the interest of better health and nutrition has greatly influenced industry practices. Fruits and vegetables sold under brand names and private labels usually sell Comprehensive Analytical Chemistry, Vol. 60. http://dx.doi.org/10.1016/B978-0-444-59562-1.00018-9 © 2013 Elsevier B.V. All rights reserved.
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for higher prices. Some stores sell food products with a producer’s certificate such as an electric tag, typically for higher prices. To save money, some food producers have falsified labels to disguise the origins of their products. To reduce the frequency of these mislabelling incidents, a valid traceability system to ensure the safety and high quality of food has been proposed. Regulations on food safety standards focus on quality control, process verification, labelling, and traceability. In Europe, there are many traditional agricultural products and foodstuffs produced in limited areas. Foods grown in certain geographical areas have unique flavours and qualities, reflecting multiple factors such as soil and climate. In accordance with knowledge and practices that have been passed down over the centuries, farmers have produced these foods for generations to protect and inherit their high quality. To authenticate the name and quality of such products, the European Union (EU) has established a geographical indication (GI) system. Three EU schemes known as the protected designation of origin (PDO), the protected geographical indication (PGI), and the traditional specialty guaranteed (TSG) promote and protect the quality of agricultural products and foodstuffs. This trade name will be crucial for the promotion of products with superior characteristics, the maintenance of biodiversity, increasing the incomes of farmers in return for a genuine effort to improve quality, the preservation of rural areas, and the availability of accurate information about the origin of products easily identifiable by local consumers. These EU schemes encourage diverse agricultural production, protect product names from imitation, and help consumers by giving them information concerning the specific character of foods they buy. For cereals, vegetables, and fruits, 123 and 174 products are registered with PDO and PGI, respectively. For example, Firiki Piliou (a Greek apple), Shaanxi ping guo (a Chinese apple), Pera de Lleida (a Spanish pear), and Fagioli Bianchi di Rotonda (a common Italian bean) are registered with PDO; Gonci kajszibarack (a Hungarian apricot), Lixian Ma Shan Yao (a Chinese yam), and Carota Novella di Ispica (an Italian carrot) are registered with PGI [1]. EU regulations have influenced other countries to adopt stringent regulations of the same calibre. In accordance with law, packaging must be labelled to indicate product information such as cultivar, cultivation area, and year of production. However, it remains very likely that packages continue to be incorrectly labelled, either accidentally or intentionally. Thus, there is a need for a simple analytical method for checking the authenticity of food products. The present study confirmed that DNA molecular markers are a powerful tool for revealing inconsistencies between plant genetics and labelling, as the genetic differentiation of vegetables and fruits is linked to their geographical origin. Plants specifically adapted to local environments serve as references for typical products. This important level of biodiversity can be preserved by promoting the economic sustainability of traditional production systems by adequate legal protection which, in turn, will also encourage the economic stability of populations in rural areas. Thus, DNA molecular markers are a necessary tool for protecting and distinguish premium products.
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Multi-element and multi-stable isotope analysis has also become an important tool for determining the provenance of foods [2,3] (see more detail in Chapter 3 on mineral profiles and in Chapter 4 on stable isotope analysis). Stable isotope analysis has become particularly useful in addressing authenticity problems. This method has been widely used to trace the origin of organic materials in various fields, such as geochemistry, biochemistry, archaeology, and petroleum chemistry. Generally, the isotopic compositions of plant materials reflect the isotopic compositions of source materials (e.g. CO2, H2O, NH4, and NO2) and their assimilation processes as well as growth environments. For example, the d13C of plants depends on fractionation during diffusion of CO2 into the leaves and the subsequent photosynthetic metabolism and water use efficiency, suggesting that carbon isotope discrimination is associated with wellwatered conditions. The nitrogen isotopic composition of plant materials mainly depends on soil nutrition. The oxygen isotopic composition mainly reflects that of local groundwater such as precipitation and meltwater. The sulphur isotope ratios are influenced by several factors such as fertilizers, sea spray, volcanic emissions, or lithology. The 87Sr/86Sr ratio in soils and the plants growing thereon depend on the geological age of the underlying rocks. For example, plants growing on young basalts normally have lower 87Sr/86Sr ratios (87Sr/86Sr < 0.706) than those growing on old crystalline rocks (87Sr/86Sr > 0.711). Therefore, the Sr isotopic ratios of plants can provide site-specific signatures depending on the geologic history of the area. On this basis, isotopic compositions have been used to investigate the authenticity of food materials. Trace element analysis has also been used as a tool for discrimination of cultivation areas. The trace element compositions of plants are mainly based on the geological and palaeoclimatic characteristics of the site of growth. Thus, the elemental compositions of foods may reveal their geographical origin. Recently, studies focused on improving the accuracy of discriminating the geographical origin of foods by combining isotope analysis and trace element analysis have been reported, demonstrating that these methods combined provide more information than either used by itself. In this chapter, we review the applications to the discrimination of agriculture practices and geographical origins of fruits and vegetables of analytical methods focusing on mass spectrometry, including inductively coupled plasma-mass spectrometry (ICP-MS) and isotope ratio mass spectrometry (Table 1).
2 DISCRIMINATION OF ORGANIC AND CONVENTIONAL FRUITS AND VEGETABLES Recently, concerns about food safety and environmental pollution have increased consumer interest in organic foods, leading to a substantial increase in the consumption of organic fruits and vegetables over the past 5 years. Organic food is produced using organic farming methods, meaning that no long-lasting chemical pesticides or fertilizers are sprayed on growing crops.
TABLE 1 Summary of the Relevant References Relating to Determination of Organic Farming or Geographical Origin in Fruits and Vegetables Agricultural Products Fruits
Purpose
Country
Parameters 15
Techniques
References
Orange, peach, and strawberry
Organic
Italy
d N (proteins of pulp)
IRMS
[5]
Citrus fruits
Organic
Italy
d15N, d13C, dD, d34S, and d18O
IRMS
[4,5]
Bilberries
Geographical origin
Denmark, Sweden, Finland, and Germany
Anthocyanidin concentrations
HPLC
[6]
Apricots
Geographical origin
Italy
DNA molecular characterization
AFLPs and SSRs
[7]
Orange juice
Geographical origin
North and South America, Africa, and Europe
dD, d13C, d15N, d34S, and
IRMS
[8]
Apple
Geographical origin
Romania
dD and d18O (water), d13C (pulp)
IRMS
[9]
Apple
Geographical origin
China and Japan
d13C and d18O
IRMS
[22]
Clementines (citrus fruits)
Geographical origin
Italy
Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Cu, Zn, Ga, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Er, and Pb in peel
ICP-MS
[10]
ICP-OES
[11]
87
Sr/86Sr
Li, Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Co, Cu, Zn, Ga, Se, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, and Er in juice Cherry
Geographical origin
Spain
Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na. Ni, Pb, Se, Sr, Ti, and Te in cherry stones Al, As, B, Ba, Be, Bi, Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mo, Na, Ni, Pb, Se, Sr, Ti, Tl, and V in the edible parts
Vegetables
Tomato, lettuce, and carrot
Organic
United Kingdom (with a few EU samples)
d15N
IRMS
[12]
Tomato, pea, broccoli, cucumber, zucchini, pumpkin, eggplant, potatoes, and corn
Organic
New Zealand
d15N
IRMS
[13]
Eggplant
Geographical origin
Spain
Morphological characteristics
[14]
DNA molecular characterization
AFLPs and SSRs
Garlic
Geographical origin
Argentina, Canada, Chile, Korea, Mexico, Pakistan, Thailand, United States, and Vietnam
Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Mo, Cd, and Ba
ICP-MS
[23]
Potato
Geographical origin
Idaho-labelled potatoes
K, Mg, Ca, Sr, Ba, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, S, Cd, Pb, and P
ICP-MS
[15]
Tomato and tomato paste
Geographical origin
Italy (Calabria, Basilicata, Puglia, and Emilia Romagna) in tomatoes, and Italy, California, China, and Greece in tomato paste
Al, As, Ba, Be, Ca, Cd, Ce, Cu, Dy, Fe, K, La, Lu, Mg, Mn, Na, Nd, Pb, Rb, Sm, Sr, Th, U, V, and Zn
ICP-MS
[16]
Onion
Geographical origin
Japan (Hokkaido, Hyogo, and Saga), China, United States, New Zealand, Thailand, Australia, and Chile
Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba
ICP-MS
[17]
Bamboo shoots and eddoe
Geographical origin
China and Japan
d13C and d18O
IRMS
[18]
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Land must also be farmed organically for 2 years before crops may be labelled as organic. In line with the EC Regulation (No. 2092/91) on organic farming, organic products are subject to controls by an accreditation and certification system. Organic foods account for about 1–2% of worldwide food sales and usually sell for higher prices than the same goods produced conventionally. Generally, the analytical controls performed on organic fruits and vegetables focus on detecting pesticide residues. Recently, the determination of stable nitrogen isotope ratios has been employed in the discrimination of organic foods [4,5,12,13,19,20]. Generally, organic fertilizers such as cow, chicken, and pig manure have higher d15N values than do chemical fertilizers. The nitrogen isotopic composition of plants is thought to depend mainly on the soil nutrition where plants are cultivated. In a previous study on the variation of d15N values in plants supplied with chemical or organic fertilizers (cow, chicken, and pig manure), the former decreased and the latter increased the d15N values of plants. Thus, the determination of d15N values is potentially useful for discrimination of organic and conventionally grown products (Figure 1) [19,20]. Bateman et al. [12] have reported the d15N values of commercially produced organic and conventionally grown tomatoes, lettuce, and carrots in order to examine the differences in the nitrogen isotope compositions of organic and conventional products. Although the nitrogen isotope values for organic and conventionally grown tomatoes and lettuce overlapped, these products had relatively higher nitrogen isotope values than did their conventional counterparts. However, there was no significant difference between the mean nitrogen isotope values for organic and conventionally grown carrots. Tomatoes and lettuce have a higher nitrogen requirement than do carrots, suggesting that the nitrogen isotope approach is capable of providing information d15N values of fertilizers Chemical fertilizers : -5 to 0‰ Organic fertilizers : +10 to +20‰ (e.g. cow, chicken, and pig manure) -15
-10
-5
0 (‰)
5
10
15
Soil Chemical fertilizers
Organic fertilizers (e.g. cow, chicken, and pig manure)
FIGURE 1 The d15N values of chemical and organic fertilizers [19,20].
20
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as to whether synthetic nitrogen fertilizers are likely to have been applied to certain crop types. Rogers [13] reported d15N values of nine different types of organic vegetables (tomatoes, peas, broccoli, cucumber, zucchini, pumpkin, eggplant, potatoes, and corn) and their conventionally grown equivalents in New Zealand. Faster growing vegetables (such as tomatoes, peas, broccoli, cucumber, and zucchini, harvested at maturity of <80 days) were compared to slower growing vegetables (such as pumpkin, eggplant, potatoes, and corn, harvested at maturity of >80 days). The faster growing vegetables exhibited the biggest isotopic difference, while slower growing crops such as eggplant (90–100 days to harvest), corn, and pumpkin (100–120 days to harvest) had the smallest isotopic values. This study demonstrates that stable nitrogen isotopes can be used as a rapid, low-cost screening tool to identify the organic growing regimen of vegetables, especially those with fast growth rates of <80 days, from their conventionally grown counterparts. For slower growing organic produce, this technique does not distinguish between the two growing regiments with as much certainty. Rapisarda et al. [4] reported the ratio of stable nitrogen isotopes in the proteins of pulp (d15NPP) to investigate the difference between organic and conventionally grown oranges, peaches, and strawberries. Organic peaches and oranges had statistically higher d15NPP values compared with conventional fruits. Therefore, the application of organic fertilizers, which notoriously increase the level of 15N in the soil, also leads to increased d15NPP values in the organic fruit. Regarding the 15N level found in strawberry pulp, no difference was observed between organic and conventional fruits because organic fertilizers are often employed even in conventional systems. Rapisarda et al. [5] reported the d15N, d13C, dD, d34S, and d18O values of citrus fruits in order to discriminate between those that were organic and those conventionally grown. The type of fertilizer used did not affect the d13C, dD, d34S, and d18O values determined for the fruit. However, the d15N values of oranges grown using fertilizer of animal origin as well as from vegetable compost were statistically higher than those grown with mineral fertilizer. Therefore, d15N values can potentially be used as an indicator to discriminate between organic and conventional citrus fruits. Organic fertilizers of different origins increase the natural abundance of 15N in organic citrus fruit, causing the d15N values to be higher than in conventional fruit.
3
TRACING THE GEOGRAPHICAL ORIGIN OF FRUITS
The concentration and composition of anthocyanins in small berries are influenced by genotype, environmental factors such as temperature, light qual˚ kerstro¨m et al. [6] ity, nutrient availability, and the timing of harvest. A analyzed anthocyanidin concentrations in Vaccinium myrtillus fruits using high-performance liquid chromatography to identify the effect of growth location and origin of parental plants. The results indicated that anthocyanidin
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concentrations in bilberries were influenced by both climatic factors and genetic control. They analyzed anthocyanidin concentrations in bilberries from wild populations growing at different latitudes. Bilberries grown at northern latitudes contained higher anthocyanidin concentrations, finding that anthocyanidin concentrations measured in 2008 were higher than those from 2007. In addition, they analyzed bilberry anthocyanidin concentrations in cloned plants grown at a single experimental site. Compilation of all the data revealed that bilberries from plants of more northerly origin had higher anthocyanidin concentrations. The most northern Finnish clone (Utsjoki) had higher anthocyanidin levels than all other clones, and anthocyanidin levels in the clones from Germany (Kiel) and southern Finland (Lapinja¨rvi) were lower than all the others. For apricots, the region of cultivation and cultivar is the important factors that influence the levels of carotenoids, which are usually higher in the Mediterranean region. Vesuvian apricots have superior characteristics such as higher sugar content, uniform colour, proper texture, pulp yield, and a rich flavour. Although several apricot cultivars have been described in the Vesuvian area, only 11 are acceptable for the production of PGI-certified fruit. These traditional cultivars result from selections by local peasants are particularly adapted to the Vesuvian environment and probably represent a distinct genetic pool [7]. This would be consistent with evidence that the genetic differentiation of apricots is linked to geographical origin. Using seven simple sequence repeats (SSRs) and four amplified fragment length polymorphisms (AFLPs) primer pairs, they reported on the molecular characterization of 36 traditional apricot (Prunus armeniaca) varieties from southern Italy, including all the varieties approved for the Albicocca Vesuviana (Vesuvian Apricot) PGI label. Cluster analysis, based on genetic distances, clearly differentiated the 11 PGI cultivars from the other genotypes. Nonetheless, among the 11 PGI cultivars, two pairs were found to have identical SSR and AFLP profiles. In addition, molecular analysis indicated the presence of mislabelling and erroneous denominations of trees from the PGI area. Rummel et al. [8] reported the dD, d13C, d15N, d34S, and 87Sr/86Sr values from the pulp of 150 authenticated orange juice samples from several regions in North and South America, Africa, and Europe. Spanish samples were easily separated from those originating in Florida, Mexico, and Cuba by analyzing a combination of 87Sr/86Sr with dD values; Brazilian samples were separated from Spanish samples by analyzing a combination of 7Sr/86Sr, d34S, and dD values. South African samples could be separated from almost all others by evaluating the Sr isotope ratio alone. However, in other samples, a certain degree of overlap existed between these single origins. The discriminant analysis classified samples into three very broad groups: Mediterranean countries (Greece, Italy, Morocco, and Spain), South America (Argentina, Brazil, Paraguay, and Uruguay), and Central America (Belize, Cuba, and Mexico) plus Florida. The most discriminatory parameters of Function 1 and Function 2 were the dD values and Sr isotope ratios,
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respectively. In the cross-validation test, performed by the software for every sample (in which each sample was excluded and treated as unknown), 88.5% were classified correctly. This kind of evaluation can also be used to distinguish subgroups and has been done within European regions. Magdas and Puscas [9] reported the dD and d18O values of water from juice and the d13C values from pulp of 45 Romanian single strength fruit juices (apples, pears, plums, and grapes) to be used for adulteration and authenticity testing. The plot of the dD–d18O values for the fruits and for the local meteoric water indicated the presence of strong evaporation in fruits. The degree of isotopic enrichment depended on the fruit species, even for those grown in the same geographic area. The obtained slopes for the fruit samples examined varied between 1.09 (pears) and 4.35 (plums), indicating that the most significant evaporation occurred for the pear samples. The isotopic values (d18O ¼ 4.5% and dD ¼ 46.4%) for water from apple juices collected in 2010 were lower than the reported values collected in previous years, reflecting the specific meteorological conditions of the year 2010 (low temperature and high humidity). The d13C mean values of apple pulp samples collected from different Transylvanian areas exhibited slight differences, probably because of the environmental conditions to which the plants were exposed. The mean value of d13C for pulp from apple juices was about 28.6%, varying between 30.7 (Salaj area) and 26.1 (Alba area). Clementines (Citrus clementina Hort. ex Tan.) are one of the most important cultivated varieties of citrus mandarins in the Mediterranean basin. Clementines grown in specific areas of Calabria, a region located in southern Italy, were recently registered as PGI from the EU. Benabdelkamel et al. [10] reported 54 PGI brand Clementine of Calabria samples and non-PGI Clementine of Calabria samples from Spain, Tunisia, and Algeria using ICP-MS analysis. The concentrations of Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Cu, Zn, Ga, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Er, and Pb in peel samples and the concentrations of Li, Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Co, Cu, Zn, Ga, Se, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, and Er in juice samples were submitted to statistical analysis. Stepwise linear discriminant analysis (S-LDA), soft independent modelling of class analogy (SIMCA), and partial least-squares discriminant analysis (PLS-DA) were used to build chemometric models able to predict PGI Clementines from those of different origins. In particular, all the chemometric approaches considered demonstrated better prediction abilities when the multi-element distribution of peel samples was included. S-LDA and SIMCA for juice samples were capable of predicting 96.6% and 100% of sample origin, respectively, whereas the PLS-DA model erroneously predicted only two samples of an independent test set. On the other hand, excellent results were achieved for peel samples by S-LDA and SIMCA models (all samples correctly classified), whereas the PLS-DA model erroneously assigned only one sample. Alicante’s Mountain Cherries are registered as PGI according to European Union Legislation. Matos-Reyes et al. [11] reported the analysis of 28 Spanish
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cherries from different areas of Spain—Arago´n, Ca´ceres, Castello´n, Huesca, and Alicante’s Mountain PGI—by ICP-OES. The concentrations of Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na. Ni, Pb, Se, Sr, Ti, and Te in cherry stones and the concentrations of Al, As, B, Ba, Be, Bi, Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mo, Na, Ni, Pb, Se, Sr, Ti, Tl, and V in the edible parts were used for chemometric analysis. The LDA of mineral concentration data for both portions showed that two or three main canonical functions were sufficient to explain most of the variability between samples taken from different origins. From the edible portion, Huesca samples were easily discriminated from the others, and Alicante’s Mountain samples were quite different from those produced in Arago´n, Ca´ceres, and Castello´n. From the cherry stones, the mineral profile made a clear differentiation between Huesca and Ca´ceres samples from the others. However, Castello´n and Arago´n samples displayed very similar distributions, as was found in the comparison of samples taken from Arago´n and Alicante. The best discrimination was found using the mineral composition of the edible portion, which permitted the correct classification of 100% of the samples examined. Nakashita and Suzuki [21] determined the carbon and oxygen bulk material isotope ratios in apple juice from Australia, China, Japan, and South Africa to discriminate their geographical origin. The apple juice from Japan was characterized by lower d13C and d18O values than those from other countries. In particular, the apple juice from China exhibited relatively high d13C values. Suzuki et al. [22] also determined the d13C and d18O values of apples from Japan and China and obtained the same characteristic results as for apple juice (Figure 2). Thus, the d13C and d18O values of bulk materials in apple juice are potentially useful for discrimination of the geographical origin of both the juice and the apples between Japan and China. The d13C values of plant organic matter have been correlated with the amount of precipitation. The d13C of plants depends on fractionation during diffusion of CO2 into the leaves and subsequent photosynthetic metabolism and water use efficiency, suggesting that carbon isotope discrimination is associated with well-watered conditions. Based on the climate statistics from the Japan Meteorological Agency, the amount of precipitation ranged from 800 to 1300 mm/year in Japan, compared to only 100 to 800 mm/year in China. These data suggest that the lower amount of precipitation in China may be reflected in the high d13C values of Chinese apples. However, there was some overlap in the distribution of d13C and d18O values between fruit originating from China and Japan.
4 TRACING GEOGRAPHICAL ORIGIN OF VEGETABLES The Almagro eggplant (Solanum melongena L.) is the only eggplant officially protected in Europe. It is used for making pickles and is produced from the local landrace traditionally grown and conserved by the farmers in the Campo de Calatrava region (situated in the province of Ciudad Real in the
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Japan
China
471
Australia
35
d18OVSMOW (‰)
30
Australia
25
China
20 Japan 15 -32
-30
-28
-26
-24
-22
-20
d13CVPDB (‰) FIGURE 2 The d13C and d18O values of Australian, Chinese, and Japanese apples [7].
centre of Spain). After harvesting, the fruits are subjected to a traditional process involving manual removal of the prickles from the fruit calyx, grading of fruit by size, boiling, natural fermentation for several days, and finally canning in a brine solution. In recent years, eggplant varieties used for pickling have been introduced from the neighbouring region of Andalusia, situated in the south of Spain. The processed Andalusian eggplants have a soft internal texture that results in a lower quality of the final product. Munoz-Falco´n et al., [14] reported the morphological characteristics of the Almagro and Andalusian eggplants as well as the hybrid fruit and looked for relevant traits for distinguishing them. Morphological characterizations were used to describe the phenotypic characteristics of commercial varieties as well as protected local landraces. No overlap in the ranges of variation was found between Almagro and Andalusian accessions for three traits (fruit pedicel width at the proximal end, fruit calyx length, and fruit calyx prickles). Moreover, molecular characterization using AFLPs and SSRs was also performed on Almagro and Andalusian varieties in order to study their diversity and relationship and for discriminating between them. Molecular markers represent an additional tool for varietal fingerprinting, for discrimination among genetically similar varieties, and for identifying hybrids. AFLP characterization showed that Almagro and Andalusian accessions are genetically diverse and share a common genetic background. In contrast, SSR made a clear distinction
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between the Almagro and Andalusian varieties, as two SSRs found were alleles exclusive to Almagro eggplant and universal to all Almagro accessions. U.S. Customs and Border Protection (CBP) implemented a special 60-day Garlic Intervention in 2002, targeting a percentage of fresh garlic imports [23]. CBP analyzed 18 elements including Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Mo, Cd, and Ba of 134 garlic samples using high resolution ICP-MS. The trace metal profile of the samples was compared to a database of authentic reference samples from Argentine, Canada, Chile, Korea, Mexico, Pakistan, Thailand, United States, and Vietnam. They found that approximately 40% of the 134 samples tested during the intervention were from a country other than the one claimed on entry documents. Moreover, CBP demonstrated that the differences in garlic varieties were less pronounced than the differences between garlic samples produced in different countries. For example, the trace metal profile of Chinese seed garlic grown in the United States matched the profile for U.S. garlic. In addition, Chinese seed garlic grown in Mexican soil for approximately 30 days produced a trace metal profile that matched the profile for Mexican garlic. Thus, the trace metal data from a sample can easily determine if it originated from the country reported. Anderson et al. [15] reported the geographical authenticity of Idaholabelled potatoes using elemental analysis (K, Mg, Ca, Sr, Ba, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, S, Cd, Pb, and P). Potato samples (608 in total) were collected from known geographic growing sites in the United States and Canada. Idaho potatoes had higher concentrations of Ca, Cd, Mg, Ni, Pb, S, and Sr than non-Idaho potatoes, while concentrations of Cu, Fe, Mn, and Zn in Idaho were higher in non-Idaho potatoes. The concentrations of Co, K, Mo, P, and V in the two groups were not significantly different. The final statistical model using 14 elements (Ba, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, P, S, Sr, and Zn) classified 330 Idaho (97%) and 251 non-Idaho (95%) samples correctly in cross-validation testing. Feudo et al. [16] reported the concentration of 32 elements (Al, As, Ba, Be, Ca, Cd, Ce, Cu, Dy, Fe, K, La, Lu, Mg, Mn, Na, Nd, Pb, Rb, Sm, Sr, Th, U, V, and Zn) in tomatoes in order to classify four Italian cultivation areas (Calabria, Basilicata, Puglia, and Emilia Romagna) and triple concentrated tomato paste samples coming from Italy and from foreign countries (California, China, and Greece). The two alkaline metals (Li and Rb) were the most important variables in the distinction of geographical origin in the discriminating functions. The origin of tomatoes and whether the areas of production of the triple concentrated pastes were inside or outside of Italy were evaluated by three supervised patterns: linear discriminant analysis (LDA), SIMCA, and K-nearest neighbours (KNN). In this case, excellent results were achieved by all models and, in particular, the KNN method correctly classified all samples as originating either from Italy or from outside Italy. Another significant achievement of the proposed method was that the classification was unaffected by the production year for the concentrated paste and the harvesting years for fresh tomatoes.
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Ariyama et al. [17] reported a scientific technique for discriminating onions (Allium cepa L.) collected from Japan (Hokkaido, Hyogo, and Saga), China, United States, New Zealand, Thailand, Australia, and Chile using mineral analysis and LDA. Of 309 samples, 108 were from Hokkaido, 52 were from Saga, 77 were from Hyogo, and 72 were from abroad. The models established by LDA using 14 elements (Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba) were used to discriminate the geographic origin between Hokkaido and abroad, Hyogo and abroad, and Saga and abroad. The discrimination accuracies obtained by cross-validation between Hokkaido and abroad were 100% and 86%, respectively; those between Hyogo and abroad were 100% and 90% accurate, respectively; and those between Saga and abroad were 98% and 90% accurate, respectively. In addition, it was demonstrated that the elemental fingerprint from a specific production area, which can be read in the crops grown therein, did not significantly change with variations in fertilization, crop year, variety, soil type, and production year if appropriate elements were chosen. Japanese and Chinese vegetables such as garlic, bamboo shoots, and eddoe (Colocasia esculenta) were analyzed to discriminate their geographical origins. Kadokura and Ariyama [24] reported the trace element composition of Japanese and Chinese garlic (Allium sativum L.), finding a significant difference in the concentrations of 12 elements (Li, Na, Mg, P, K, Ca, Mn, Fe, Cu, Sr, Mo, and Cd) between the two. The garlic samples were separated according to the amounts of five elements (Li, Na, Mn, Cu, and Zn) by LDA to categorize them into particular groups. All garlic samples (79 samples) were correctly classified in 10-fold cross-validation. Suzuki and Nakashita [18] reported the carbon and oxygen isotope ratios of the bamboo shoots and eddoe; the d13C values of Japanese bamboo shoots and eddoe were relatively lower than those of their Chinese counterparts, while there was no significant difference in d18O between Chinese and Japanese bamboo shoots and eddoe (Figure 3). Moreover, the distribution of d13C and d18O values for Japanese and Chinese bamboo shoots and eddoe overlapped one another. Because China is one of the largest countries in the world, the distribution of stable isotope ratios in China is expected to be quite large. Specifically, northern China is similar to Japan in geological characteristics such as latitude. Therefore, it is very difficult to clearly discriminate the geographical origin of food products from China and Japan.
5 FUTURE PERSPECTIVES IN PDO AUTHENTICATION OF FRUITS AND VEGETABLES The combined use of multiple analytical methods is needed to improve the accuracy of discrimination of the geographical origin of fruits and vegetables. We determined stable isotope ratios and trace element compositions of bulk materials in apples from various cultivation areas in China and Japan to
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B China
Japan
Japan
35
China
30 China
d18OVSMOW (‰)
d18OVSMOW (‰)
30
25
20
15 -30
25 China Japan 20
Japan
-29
-28
-27
-26
-25
-24
15 -30
-28
-26
13
d CVPDB (‰)
-24
-22
d13CVPDB (‰)
FIGURE 3 The d13C and d18O values of Chinese and Japanese bamboo shoots (A), and eddoe (B) [22].
A
B JPN (Aomori)
JPN (Nagano)
CHN
JPN (Aomori)
35
JPN (Nagano)
Japan (Aomori)
China
China 3 2
Function 2
d18OVSMOW (‰)
30
25
Japan (Aomori)
20
15 −30
CHN
4
−26
−24
−22
0 −8
−6
−4
−2
0
2
4
6
8
−1 −2
Japan (Nagano) −28
1
−3
Japan (Nagano)
−4
−20
Function 1
13
d CVPDB (‰) 13
18
FIGURE 4 Distributions of the d C and d O values of Chinese, Japanese (Aomori and Nagano) apples (A) and the corresponding dendrogram using the d13C and d18O values and nine elements (Mg, Ca, Mn, Fe, Ni, Cu, Zn, Ga, As, Rb, Sr, Mo, Cd, Cs, Ba, Tl, and Pb) of them (B).
discriminate their geographical origin (Figure 4). We determined stable isotope ratios and trace element compositions of apples from various cultivated areas in China and Japan to discriminate their geographical origin. Of 188 samples, 98 were from Aomori Pref. (Japan), 42 were from Nagano Pref. (Japan), and 48 were from China. The d13C and d18O values of Chinese apples were significantly higher than those of Japanese apples. However, there was no significant difference between Aomori and Nagano Pref. Using the analytical results of d13C and d18O values and 18 elements (Mg, Ca, Mn, Fe, Co, Ni,
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Cu, Zn, Ga, As, Rb, Sr, Mo, Cd, Cs, Ba, Tl, and Pb), apple samples were analyzed by cluster analysis and canonical discriminant analysis to categorize them into specific groups. As a result, the apple samples were divided into three groups: China, Aomori Pref. (Japan), and Nagano Pref. (Japan). Thus, stable isotope and trace element analysis are potentially useful for discriminating the geographical origin of Chinese and Japanese apples [25]. The previous studies of food authenticity introduced in this chapter reported the analysis of raw foods. However, many fruits and vegetables are also processed into juice and jam or are preserved in syrup, frozen, and canned. Such processed products are also targets of mislabelling. Therefore, analytical methods are required for discrimination of food authenticity of processed products as well as fresh fruits and vegetables. However, processed products include seasonings such as salt, sugar, and spices, which contain trace and light elements. Moreover, they are cooked using various food-processing processes such as cutting, boiling, and grilling, factors which can influence the stable isotope ratios and mineral compositions of the processed products. For example, Fung et al. [26] reported the statistical analysis of experimental data on raw versus frozen spinach. There were significant differences at the 1% level between the calcium, chloride, iron, manganese, phosphorus, potassium, and sodium content and a difference at the 5% level between the magnesium content of raw and frozen spinach. The loss of essential elements in commercial freezing of vegetables is probably due to leaching into the blanching and cooling waters. The differences in retention of elements in different frozen vegetables probably depend upon the different chemical forms of the element in the vegetable tissues and the solubility of these compounds in water. The pre-blanching and blanching processes may damage the vegetable cell membranes, which are through to allow free diffusion of ions between the two systems depending upon time of exposure, degree of maceration, or tissue thickness. In one case, boiled bamboo shoots were subjected to multi-trace elemental analysis to discriminate the geographical origin, but no significant difference was found between Japanese and Chinese boiled bamboo shoots because the trace elements were depleted during processing steps such as washing and boiling. On the other hand, carbon, nitrogen, oxygen, and hydrogen are the main elements from which our bodies are composed and are not vulnerable to food-processing operations. Therefore, although pre-treatment for removing seasoning is required, stable isotope analysis is expected to be a potential tool for discrimination of geographical origin for processed fruits and vegetables.
REFERENCES [1] European Commission. Agriculture and rural development. Quality policy. http://ec.europa. eu/agriculture/quality/. [2] Kelly S, Heaton K, Hoogewerff J. Tracing the geographical origin of food: the application of multi-element and multi-isotope analysis. Trends Food Sci Technol 2005;16:555.
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[3] Gonzalvez A, de la Guardia M. Trace-element composition and stable-isotope ratio for discrimination of foods with Protected Designation of Origin. Trends Anal Chem 2009;28:1295. [4] Rapisarda P, Camin F, Fabroni S, Perini M, Torrisi B, Intrigliolo F. Influence of different organic fertilizers on quality parameters and the d15N, d13C, d2H, d34S, and d18O values of orange fruit (Citrus sinensis L. Osbeck). J Agric Food Chem 2010;58:3502. [5] Rapisarda P, Camin F, Faedi W, Paoletti F, Tabilio MR. New markers for the traceability of organic fruit. Acta Horticul 2010;873:173. ˚ kerstro¨m A, Jaakola L, Ba˚ng U, Ja¨derlund A. Effects of latitude-related factors and geo[6] A graphical origin on anthocyanidin concentrations in fruits of Vaccinium myrtillus L. (Bilberries). J Agric Food Chem 2010;58:11939. [7] Rao R, Bencivenni M, La Mura M, Araujo-Burgos T, Corrado G. Molecular characterisation of Vesuvian apricot cultivars: implications for the certification and authentication of protected plant material. J Hortic Sci Biotechnol 2010;85:42. [8] Rummel S, Hoelzl S, Horn P, Rossmann A, Schlicht C. The combination of stable isotope abundance ratios of H, C, N and S with 87Sr/86Sr for geographical origin assignment of orange juices. Food Chem 2010;118:890. [9] Magdas DA, Puscas R. Stable isotopes determination in some Romanian fruit juices. Isotopes Environ Health Stud 2011;47:372. [10] Benabdelkamel H, Di Donna L, Mazzotti F, Naccarato A, Sindona G, Tagarelli A, et al. Authenticity of PGI “Clementine of Calabria” by multielement fingerprint. J Agric Food Chem 2012;60:3717. [11] Matos-Reyes MN, Simonot J, Lo´pez-Salazar O, Cervera ML, de la Guardia M. Authentication of Alicante’s mountain cherries protected designation of origin by their mineral profile. Food Chem. in press. [12] Bateman AS, Kelly SD, Woolfe M. Nitrogen isotope composition of organically and conventionally grown crops. J Agric Food Chem 2007;55:2664. [13] Rogers KM. Nitrogen isotopes as a screening tool to determine the growing regimen of some organic and nonorganic supermarket produce from New Zealand. J Agric Food Chem 2008;56:4078. [14] Mu noz-Falco´n J, Prohens J, Vilanova S, Ribas F, Castroc A, Nueza F. Distinguishing a protected geographical indication vegetable (Almagro eggplant) from closely related varieties with selected morphological traits and molecular markers. J Sci Food Agric 2009;89:320. [15] Anderson KA, Magnuson BA, Tschirgi ML, Smith B. Determining the geographic origin of potatoes with trace metal analysis using statistical and neural network classifiers. J Agric Food Chem 1999;47:1568. [16] Feudo GL, Naccarato A, Sindona G, Tagarelli A. Investigating the origin of tomatoes and triple concentrated tomato pastes through multielement determination by inductively coupled plasma mass Spectrometry and statistical analysis. J Agric Food Chem 2010;58:3801. [17] Ariyama K, Aoyama Y, Mochizuki A, Homura Y, Kadokura M, Yasui A. Determination of the geographic origin of onions between three main production areas in Japan and other countries by mineral composition. J Agric Food Chem 2007;55:347. [18] Suzuki Y, Nakashita R. Approach to the food authenticity problems by using multi-element stable isotope ratio analysis. Kagaku to Seibutsu 2010;4(8):121 [in Japanese]. [19] Kohl DH, Shearer GB, Commones B. Variation of 15N in corn and soil following applications of fertilizer nitrogen. Soil Sci Soc Am J 1973;37:888. [20] Meints VW, Shearer G, Kohl DH, Kurtz LT. A comparison of unenriched vs. 15N enriched fertilizer as a tracer for N fertilizer uptake. Soil Sci 1975;119:421.
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[21] Nakashita R, Suzuki Y. Stable isotope analysis as a potential index for verifying the geographical origin of apple juice. Bunseki Kagaku 2009;5(8):1059 [in Japanese]. [22] Suzuki Y, Nakashita R, Kobe R, Kitai A, Tomiyama S. Tracing the geographical origin of Japanese (Aomori Prefecture) and Chinese apples using stable carbon and oxygen isotope analyses. Nippon Shokuhin Kagaku Kogaku Kaishi 2012;5(9):69 [in Japanese]. [23] Smith RG. Determination of the country of origin of garlic (Allium sativum) using trace metal profiling. J Agric Food Chem 2010;58:3801. [24] Kadokura M, Ariyama K. Development of technique for estimation of geographical origin of Japanese and Chinese garlic using trace element analyses. Food and Agricultural Materials Inspection Center research report. No. 3101; 2007 [in Japanese]. [25] Suzuki Y, Takeuchi J, Nakashita R, Kobe R, Watanabe I. Tracing the geographical origin of Chinese and Japanese apple using stable carbon and oxygen isotope analysis and trace element analysis. In: Abstract of 5th international symposium on recent advances in food analysis, Prague, November 1–4. 2011. [26] Fung AC, Lopez A, Cooler FW. Essential elements in fresh and in frozen spinach and collards. J Food Sci 1978;43:897.
Authentication and Traceability of Fruits and Vegetables Yaeko Suzuki* and Rumiko Nakashita{ *
Food Analysis Laboratory, Analytical Science Division, National Food Research Institute, National Agriculture and Food Research Organization (NARO), Kannondai, Tsukuba, Ibaraki, Japan { Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan
Chapter Outline 1. The Geographical Indication System of Fruits and Vegetables Around the World 461 2. Discrimination of Organic and Conventional Fruits and Vegetables 463
3. Tracing the Geographical Origin of Fruits 4. Tracing Geographical Origin of Vegetables 5. Future Perspectives in PDO Authentication of Fruits and Vegetables References
467 470
473 475
1 THE GEOGRAPHICAL INDICATION SYSTEM OF FRUITS AND VEGETABLES AROUND THE WORLD Fruit and vegetables play an important role in a healthy, balanced diet. They contain vitamins, minerals, fibre, sugar, as well as other minor nutrients. The types and amounts of available fruits and vegetables have been expanding globally, and consumers can now obtain products from all over the world. However, in recent years, consumers have experienced numerous negative incidents such as unsafe levels of residual pesticide in exported fruits and vegetables, which has prompted greater awareness of geographical origin and chemical levels in food. Such concerned consumers prefer to buy fruits and vegetables directly from farmers even if the products are more expensive than exported equivalents. The increased demand for reliable food in the interest of better health and nutrition has greatly influenced industry practices. Fruits and vegetables sold under brand names and private labels usually sell Comprehensive Analytical Chemistry, Vol. 60. http://dx.doi.org/10.1016/B978-0-444-59562-1.00018-9 © 2013 Elsevier B.V. All rights reserved.
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for higher prices. Some stores sell food products with a producer’s certificate such as an electric tag, typically for higher prices. To save money, some food producers have falsified labels to disguise the origins of their products. To reduce the frequency of these mislabelling incidents, a valid traceability system to ensure the safety and high quality of food has been proposed. Regulations on food safety standards focus on quality control, process verification, labelling, and traceability. In Europe, there are many traditional agricultural products and foodstuffs produced in limited areas. Foods grown in certain geographical areas have unique flavours and qualities, reflecting multiple factors such as soil and climate. In accordance with knowledge and practices that have been passed down over the centuries, farmers have produced these foods for generations to protect and inherit their high quality. To authenticate the name and quality of such products, the European Union (EU) has established a geographical indication (GI) system. Three EU schemes known as the protected designation of origin (PDO), the protected geographical indication (PGI), and the traditional specialty guaranteed (TSG) promote and protect the quality of agricultural products and foodstuffs. This trade name will be crucial for the promotion of products with superior characteristics, the maintenance of biodiversity, increasing the incomes of farmers in return for a genuine effort to improve quality, the preservation of rural areas, and the availability of accurate information about the origin of products easily identifiable by local consumers. These EU schemes encourage diverse agricultural production, protect product names from imitation, and help consumers by giving them information concerning the specific character of foods they buy. For cereals, vegetables, and fruits, 123 and 174 products are registered with PDO and PGI, respectively. For example, Firiki Piliou (a Greek apple), Shaanxi ping guo (a Chinese apple), Pera de Lleida (a Spanish pear), and Fagioli Bianchi di Rotonda (a common Italian bean) are registered with PDO; Gonci kajszibarack (a Hungarian apricot), Lixian Ma Shan Yao (a Chinese yam), and Carota Novella di Ispica (an Italian carrot) are registered with PGI [1]. EU regulations have influenced other countries to adopt stringent regulations of the same calibre. In accordance with law, packaging must be labelled to indicate product information such as cultivar, cultivation area, and year of production. However, it remains very likely that packages continue to be incorrectly labelled, either accidentally or intentionally. Thus, there is a need for a simple analytical method for checking the authenticity of food products. The present study confirmed that DNA molecular markers are a powerful tool for revealing inconsistencies between plant genetics and labelling, as the genetic differentiation of vegetables and fruits is linked to their geographical origin. Plants specifically adapted to local environments serve as references for typical products. This important level of biodiversity can be preserved by promoting the economic sustainability of traditional production systems by adequate legal protection which, in turn, will also encourage the economic stability of populations in rural areas. Thus, DNA molecular markers are a necessary tool for protecting and distinguish premium products.
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Multi-element and multi-stable isotope analysis has also become an important tool for determining the provenance of foods [2,3] (see more detail in Chapter 3 on mineral profiles and in Chapter 4 on stable isotope analysis). Stable isotope analysis has become particularly useful in addressing authenticity problems. This method has been widely used to trace the origin of organic materials in various fields, such as geochemistry, biochemistry, archaeology, and petroleum chemistry. Generally, the isotopic compositions of plant materials reflect the isotopic compositions of source materials (e.g. CO2, H2O, NH4, and NO2) and their assimilation processes as well as growth environments. For example, the d13C of plants depends on fractionation during diffusion of CO2 into the leaves and the subsequent photosynthetic metabolism and water use efficiency, suggesting that carbon isotope discrimination is associated with wellwatered conditions. The nitrogen isotopic composition of plant materials mainly depends on soil nutrition. The oxygen isotopic composition mainly reflects that of local groundwater such as precipitation and meltwater. The sulphur isotope ratios are influenced by several factors such as fertilizers, sea spray, volcanic emissions, or lithology. The 87Sr/86Sr ratio in soils and the plants growing thereon depend on the geological age of the underlying rocks. For example, plants growing on young basalts normally have lower 87Sr/86Sr ratios (87Sr/86Sr < 0.706) than those growing on old crystalline rocks (87Sr/86Sr > 0.711). Therefore, the Sr isotopic ratios of plants can provide site-specific signatures depending on the geologic history of the area. On this basis, isotopic compositions have been used to investigate the authenticity of food materials. Trace element analysis has also been used as a tool for discrimination of cultivation areas. The trace element compositions of plants are mainly based on the geological and palaeoclimatic characteristics of the site of growth. Thus, the elemental compositions of foods may reveal their geographical origin. Recently, studies focused on improving the accuracy of discriminating the geographical origin of foods by combining isotope analysis and trace element analysis have been reported, demonstrating that these methods combined provide more information than either used by itself. In this chapter, we review the applications to the discrimination of agriculture practices and geographical origins of fruits and vegetables of analytical methods focusing on mass spectrometry, including inductively coupled plasma-mass spectrometry (ICP-MS) and isotope ratio mass spectrometry (Table 1).
2 DISCRIMINATION OF ORGANIC AND CONVENTIONAL FRUITS AND VEGETABLES Recently, concerns about food safety and environmental pollution have increased consumer interest in organic foods, leading to a substantial increase in the consumption of organic fruits and vegetables over the past 5 years. Organic food is produced using organic farming methods, meaning that no long-lasting chemical pesticides or fertilizers are sprayed on growing crops.
TABLE 1 Summary of the Relevant References Relating to Determination of Organic Farming or Geographical Origin in Fruits and Vegetables Agricultural Products Fruits
Purpose
Country
Parameters 15
Techniques
References
Orange, peach, and strawberry
Organic
Italy
d N (proteins of pulp)
IRMS
[5]
Citrus fruits
Organic
Italy
d15N, d13C, dD, d34S, and d18O
IRMS
[4,5]
Bilberries
Geographical origin
Denmark, Sweden, Finland, and Germany
Anthocyanidin concentrations
HPLC
[6]
Apricots
Geographical origin
Italy
DNA molecular characterization
AFLPs and SSRs
[7]
Orange juice
Geographical origin
North and South America, Africa, and Europe
dD, d13C, d15N, d34S, and
IRMS
[8]
Apple
Geographical origin
Romania
dD and d18O (water), d13C (pulp)
IRMS
[9]
Apple
Geographical origin
China and Japan
d13C and d18O
IRMS
[22]
Clementines (citrus fruits)
Geographical origin
Italy
Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Cu, Zn, Ga, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Er, and Pb in peel
ICP-MS
[10]
ICP-OES
[11]
87
Sr/86Sr
Li, Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Co, Cu, Zn, Ga, Se, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, and Er in juice Cherry
Geographical origin
Spain
Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na. Ni, Pb, Se, Sr, Ti, and Te in cherry stones Al, As, B, Ba, Be, Bi, Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mo, Na, Ni, Pb, Se, Sr, Ti, Tl, and V in the edible parts
Vegetables
Tomato, lettuce, and carrot
Organic
United Kingdom (with a few EU samples)
d15N
IRMS
[12]
Tomato, pea, broccoli, cucumber, zucchini, pumpkin, eggplant, potatoes, and corn
Organic
New Zealand
d15N
IRMS
[13]
Eggplant
Geographical origin
Spain
Morphological characteristics
[14]
DNA molecular characterization
AFLPs and SSRs
Garlic
Geographical origin
Argentina, Canada, Chile, Korea, Mexico, Pakistan, Thailand, United States, and Vietnam
Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Mo, Cd, and Ba
ICP-MS
[23]
Potato
Geographical origin
Idaho-labelled potatoes
K, Mg, Ca, Sr, Ba, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, S, Cd, Pb, and P
ICP-MS
[15]
Tomato and tomato paste
Geographical origin
Italy (Calabria, Basilicata, Puglia, and Emilia Romagna) in tomatoes, and Italy, California, China, and Greece in tomato paste
Al, As, Ba, Be, Ca, Cd, Ce, Cu, Dy, Fe, K, La, Lu, Mg, Mn, Na, Nd, Pb, Rb, Sm, Sr, Th, U, V, and Zn
ICP-MS
[16]
Onion
Geographical origin
Japan (Hokkaido, Hyogo, and Saga), China, United States, New Zealand, Thailand, Australia, and Chile
Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba
ICP-MS
[17]
Bamboo shoots and eddoe
Geographical origin
China and Japan
d13C and d18O
IRMS
[18]
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Land must also be farmed organically for 2 years before crops may be labelled as organic. In line with the EC Regulation (No. 2092/91) on organic farming, organic products are subject to controls by an accreditation and certification system. Organic foods account for about 1–2% of worldwide food sales and usually sell for higher prices than the same goods produced conventionally. Generally, the analytical controls performed on organic fruits and vegetables focus on detecting pesticide residues. Recently, the determination of stable nitrogen isotope ratios has been employed in the discrimination of organic foods [4,5,12,13,19,20]. Generally, organic fertilizers such as cow, chicken, and pig manure have higher d15N values than do chemical fertilizers. The nitrogen isotopic composition of plants is thought to depend mainly on the soil nutrition where plants are cultivated. In a previous study on the variation of d15N values in plants supplied with chemical or organic fertilizers (cow, chicken, and pig manure), the former decreased and the latter increased the d15N values of plants. Thus, the determination of d15N values is potentially useful for discrimination of organic and conventionally grown products (Figure 1) [19,20]. Bateman et al. [12] have reported the d15N values of commercially produced organic and conventionally grown tomatoes, lettuce, and carrots in order to examine the differences in the nitrogen isotope compositions of organic and conventional products. Although the nitrogen isotope values for organic and conventionally grown tomatoes and lettuce overlapped, these products had relatively higher nitrogen isotope values than did their conventional counterparts. However, there was no significant difference between the mean nitrogen isotope values for organic and conventionally grown carrots. Tomatoes and lettuce have a higher nitrogen requirement than do carrots, suggesting that the nitrogen isotope approach is capable of providing information d15N values of fertilizers Chemical fertilizers : -5 to 0‰ Organic fertilizers : +10 to +20‰ (e.g. cow, chicken, and pig manure) -15
-10
-5
0 (‰)
5
10
15
Soil Chemical fertilizers
Organic fertilizers (e.g. cow, chicken, and pig manure)
FIGURE 1 The d15N values of chemical and organic fertilizers [19,20].
20
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as to whether synthetic nitrogen fertilizers are likely to have been applied to certain crop types. Rogers [13] reported d15N values of nine different types of organic vegetables (tomatoes, peas, broccoli, cucumber, zucchini, pumpkin, eggplant, potatoes, and corn) and their conventionally grown equivalents in New Zealand. Faster growing vegetables (such as tomatoes, peas, broccoli, cucumber, and zucchini, harvested at maturity of <80 days) were compared to slower growing vegetables (such as pumpkin, eggplant, potatoes, and corn, harvested at maturity of >80 days). The faster growing vegetables exhibited the biggest isotopic difference, while slower growing crops such as eggplant (90–100 days to harvest), corn, and pumpkin (100–120 days to harvest) had the smallest isotopic values. This study demonstrates that stable nitrogen isotopes can be used as a rapid, low-cost screening tool to identify the organic growing regimen of vegetables, especially those with fast growth rates of <80 days, from their conventionally grown counterparts. For slower growing organic produce, this technique does not distinguish between the two growing regiments with as much certainty. Rapisarda et al. [4] reported the ratio of stable nitrogen isotopes in the proteins of pulp (d15NPP) to investigate the difference between organic and conventionally grown oranges, peaches, and strawberries. Organic peaches and oranges had statistically higher d15NPP values compared with conventional fruits. Therefore, the application of organic fertilizers, which notoriously increase the level of 15N in the soil, also leads to increased d15NPP values in the organic fruit. Regarding the 15N level found in strawberry pulp, no difference was observed between organic and conventional fruits because organic fertilizers are often employed even in conventional systems. Rapisarda et al. [5] reported the d15N, d13C, dD, d34S, and d18O values of citrus fruits in order to discriminate between those that were organic and those conventionally grown. The type of fertilizer used did not affect the d13C, dD, d34S, and d18O values determined for the fruit. However, the d15N values of oranges grown using fertilizer of animal origin as well as from vegetable compost were statistically higher than those grown with mineral fertilizer. Therefore, d15N values can potentially be used as an indicator to discriminate between organic and conventional citrus fruits. Organic fertilizers of different origins increase the natural abundance of 15N in organic citrus fruit, causing the d15N values to be higher than in conventional fruit.
3
TRACING THE GEOGRAPHICAL ORIGIN OF FRUITS
The concentration and composition of anthocyanins in small berries are influenced by genotype, environmental factors such as temperature, light qual˚ kerstro¨m et al. [6] ity, nutrient availability, and the timing of harvest. A analyzed anthocyanidin concentrations in Vaccinium myrtillus fruits using high-performance liquid chromatography to identify the effect of growth location and origin of parental plants. The results indicated that anthocyanidin
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concentrations in bilberries were influenced by both climatic factors and genetic control. They analyzed anthocyanidin concentrations in bilberries from wild populations growing at different latitudes. Bilberries grown at northern latitudes contained higher anthocyanidin concentrations, finding that anthocyanidin concentrations measured in 2008 were higher than those from 2007. In addition, they analyzed bilberry anthocyanidin concentrations in cloned plants grown at a single experimental site. Compilation of all the data revealed that bilberries from plants of more northerly origin had higher anthocyanidin concentrations. The most northern Finnish clone (Utsjoki) had higher anthocyanidin levels than all other clones, and anthocyanidin levels in the clones from Germany (Kiel) and southern Finland (Lapinja¨rvi) were lower than all the others. For apricots, the region of cultivation and cultivar is the important factors that influence the levels of carotenoids, which are usually higher in the Mediterranean region. Vesuvian apricots have superior characteristics such as higher sugar content, uniform colour, proper texture, pulp yield, and a rich flavour. Although several apricot cultivars have been described in the Vesuvian area, only 11 are acceptable for the production of PGI-certified fruit. These traditional cultivars result from selections by local peasants are particularly adapted to the Vesuvian environment and probably represent a distinct genetic pool [7]. This would be consistent with evidence that the genetic differentiation of apricots is linked to geographical origin. Using seven simple sequence repeats (SSRs) and four amplified fragment length polymorphisms (AFLPs) primer pairs, they reported on the molecular characterization of 36 traditional apricot (Prunus armeniaca) varieties from southern Italy, including all the varieties approved for the Albicocca Vesuviana (Vesuvian Apricot) PGI label. Cluster analysis, based on genetic distances, clearly differentiated the 11 PGI cultivars from the other genotypes. Nonetheless, among the 11 PGI cultivars, two pairs were found to have identical SSR and AFLP profiles. In addition, molecular analysis indicated the presence of mislabelling and erroneous denominations of trees from the PGI area. Rummel et al. [8] reported the dD, d13C, d15N, d34S, and 87Sr/86Sr values from the pulp of 150 authenticated orange juice samples from several regions in North and South America, Africa, and Europe. Spanish samples were easily separated from those originating in Florida, Mexico, and Cuba by analyzing a combination of 87Sr/86Sr with dD values; Brazilian samples were separated from Spanish samples by analyzing a combination of 7Sr/86Sr, d34S, and dD values. South African samples could be separated from almost all others by evaluating the Sr isotope ratio alone. However, in other samples, a certain degree of overlap existed between these single origins. The discriminant analysis classified samples into three very broad groups: Mediterranean countries (Greece, Italy, Morocco, and Spain), South America (Argentina, Brazil, Paraguay, and Uruguay), and Central America (Belize, Cuba, and Mexico) plus Florida. The most discriminatory parameters of Function 1 and Function 2 were the dD values and Sr isotope ratios,
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respectively. In the cross-validation test, performed by the software for every sample (in which each sample was excluded and treated as unknown), 88.5% were classified correctly. This kind of evaluation can also be used to distinguish subgroups and has been done within European regions. Magdas and Puscas [9] reported the dD and d18O values of water from juice and the d13C values from pulp of 45 Romanian single strength fruit juices (apples, pears, plums, and grapes) to be used for adulteration and authenticity testing. The plot of the dD–d18O values for the fruits and for the local meteoric water indicated the presence of strong evaporation in fruits. The degree of isotopic enrichment depended on the fruit species, even for those grown in the same geographic area. The obtained slopes for the fruit samples examined varied between 1.09 (pears) and 4.35 (plums), indicating that the most significant evaporation occurred for the pear samples. The isotopic values (d18O ¼ 4.5% and dD ¼ 46.4%) for water from apple juices collected in 2010 were lower than the reported values collected in previous years, reflecting the specific meteorological conditions of the year 2010 (low temperature and high humidity). The d13C mean values of apple pulp samples collected from different Transylvanian areas exhibited slight differences, probably because of the environmental conditions to which the plants were exposed. The mean value of d13C for pulp from apple juices was about 28.6%, varying between 30.7 (Salaj area) and 26.1 (Alba area). Clementines (Citrus clementina Hort. ex Tan.) are one of the most important cultivated varieties of citrus mandarins in the Mediterranean basin. Clementines grown in specific areas of Calabria, a region located in southern Italy, were recently registered as PGI from the EU. Benabdelkamel et al. [10] reported 54 PGI brand Clementine of Calabria samples and non-PGI Clementine of Calabria samples from Spain, Tunisia, and Algeria using ICP-MS analysis. The concentrations of Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Cu, Zn, Ga, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Er, and Pb in peel samples and the concentrations of Li, Na, Mg, Al, Ca, Cr, Fe, Mn, Ni, Co, Cu, Zn, Ga, Se, Rb, Sr, Y, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, and Er in juice samples were submitted to statistical analysis. Stepwise linear discriminant analysis (S-LDA), soft independent modelling of class analogy (SIMCA), and partial least-squares discriminant analysis (PLS-DA) were used to build chemometric models able to predict PGI Clementines from those of different origins. In particular, all the chemometric approaches considered demonstrated better prediction abilities when the multi-element distribution of peel samples was included. S-LDA and SIMCA for juice samples were capable of predicting 96.6% and 100% of sample origin, respectively, whereas the PLS-DA model erroneously predicted only two samples of an independent test set. On the other hand, excellent results were achieved for peel samples by S-LDA and SIMCA models (all samples correctly classified), whereas the PLS-DA model erroneously assigned only one sample. Alicante’s Mountain Cherries are registered as PGI according to European Union Legislation. Matos-Reyes et al. [11] reported the analysis of 28 Spanish
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cherries from different areas of Spain—Arago´n, Ca´ceres, Castello´n, Huesca, and Alicante’s Mountain PGI—by ICP-OES. The concentrations of Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na. Ni, Pb, Se, Sr, Ti, and Te in cherry stones and the concentrations of Al, As, B, Ba, Be, Bi, Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mo, Na, Ni, Pb, Se, Sr, Ti, Tl, and V in the edible parts were used for chemometric analysis. The LDA of mineral concentration data for both portions showed that two or three main canonical functions were sufficient to explain most of the variability between samples taken from different origins. From the edible portion, Huesca samples were easily discriminated from the others, and Alicante’s Mountain samples were quite different from those produced in Arago´n, Ca´ceres, and Castello´n. From the cherry stones, the mineral profile made a clear differentiation between Huesca and Ca´ceres samples from the others. However, Castello´n and Arago´n samples displayed very similar distributions, as was found in the comparison of samples taken from Arago´n and Alicante. The best discrimination was found using the mineral composition of the edible portion, which permitted the correct classification of 100% of the samples examined. Nakashita and Suzuki [21] determined the carbon and oxygen bulk material isotope ratios in apple juice from Australia, China, Japan, and South Africa to discriminate their geographical origin. The apple juice from Japan was characterized by lower d13C and d18O values than those from other countries. In particular, the apple juice from China exhibited relatively high d13C values. Suzuki et al. [22] also determined the d13C and d18O values of apples from Japan and China and obtained the same characteristic results as for apple juice (Figure 2). Thus, the d13C and d18O values of bulk materials in apple juice are potentially useful for discrimination of the geographical origin of both the juice and the apples between Japan and China. The d13C values of plant organic matter have been correlated with the amount of precipitation. The d13C of plants depends on fractionation during diffusion of CO2 into the leaves and subsequent photosynthetic metabolism and water use efficiency, suggesting that carbon isotope discrimination is associated with well-watered conditions. Based on the climate statistics from the Japan Meteorological Agency, the amount of precipitation ranged from 800 to 1300 mm/year in Japan, compared to only 100 to 800 mm/year in China. These data suggest that the lower amount of precipitation in China may be reflected in the high d13C values of Chinese apples. However, there was some overlap in the distribution of d13C and d18O values between fruit originating from China and Japan.
4 TRACING GEOGRAPHICAL ORIGIN OF VEGETABLES The Almagro eggplant (Solanum melongena L.) is the only eggplant officially protected in Europe. It is used for making pickles and is produced from the local landrace traditionally grown and conserved by the farmers in the Campo de Calatrava region (situated in the province of Ciudad Real in the
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d13CVPDB (‰) FIGURE 2 The d13C and d18O values of Australian, Chinese, and Japanese apples [7].
centre of Spain). After harvesting, the fruits are subjected to a traditional process involving manual removal of the prickles from the fruit calyx, grading of fruit by size, boiling, natural fermentation for several days, and finally canning in a brine solution. In recent years, eggplant varieties used for pickling have been introduced from the neighbouring region of Andalusia, situated in the south of Spain. The processed Andalusian eggplants have a soft internal texture that results in a lower quality of the final product. Munoz-Falco´n et al., [14] reported the morphological characteristics of the Almagro and Andalusian eggplants as well as the hybrid fruit and looked for relevant traits for distinguishing them. Morphological characterizations were used to describe the phenotypic characteristics of commercial varieties as well as protected local landraces. No overlap in the ranges of variation was found between Almagro and Andalusian accessions for three traits (fruit pedicel width at the proximal end, fruit calyx length, and fruit calyx prickles). Moreover, molecular characterization using AFLPs and SSRs was also performed on Almagro and Andalusian varieties in order to study their diversity and relationship and for discriminating between them. Molecular markers represent an additional tool for varietal fingerprinting, for discrimination among genetically similar varieties, and for identifying hybrids. AFLP characterization showed that Almagro and Andalusian accessions are genetically diverse and share a common genetic background. In contrast, SSR made a clear distinction
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between the Almagro and Andalusian varieties, as two SSRs found were alleles exclusive to Almagro eggplant and universal to all Almagro accessions. U.S. Customs and Border Protection (CBP) implemented a special 60-day Garlic Intervention in 2002, targeting a percentage of fresh garlic imports [23]. CBP analyzed 18 elements including Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Mo, Cd, and Ba of 134 garlic samples using high resolution ICP-MS. The trace metal profile of the samples was compared to a database of authentic reference samples from Argentine, Canada, Chile, Korea, Mexico, Pakistan, Thailand, United States, and Vietnam. They found that approximately 40% of the 134 samples tested during the intervention were from a country other than the one claimed on entry documents. Moreover, CBP demonstrated that the differences in garlic varieties were less pronounced than the differences between garlic samples produced in different countries. For example, the trace metal profile of Chinese seed garlic grown in the United States matched the profile for U.S. garlic. In addition, Chinese seed garlic grown in Mexican soil for approximately 30 days produced a trace metal profile that matched the profile for Mexican garlic. Thus, the trace metal data from a sample can easily determine if it originated from the country reported. Anderson et al. [15] reported the geographical authenticity of Idaholabelled potatoes using elemental analysis (K, Mg, Ca, Sr, Ba, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, S, Cd, Pb, and P). Potato samples (608 in total) were collected from known geographic growing sites in the United States and Canada. Idaho potatoes had higher concentrations of Ca, Cd, Mg, Ni, Pb, S, and Sr than non-Idaho potatoes, while concentrations of Cu, Fe, Mn, and Zn in Idaho were higher in non-Idaho potatoes. The concentrations of Co, K, Mo, P, and V in the two groups were not significantly different. The final statistical model using 14 elements (Ba, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, P, S, Sr, and Zn) classified 330 Idaho (97%) and 251 non-Idaho (95%) samples correctly in cross-validation testing. Feudo et al. [16] reported the concentration of 32 elements (Al, As, Ba, Be, Ca, Cd, Ce, Cu, Dy, Fe, K, La, Lu, Mg, Mn, Na, Nd, Pb, Rb, Sm, Sr, Th, U, V, and Zn) in tomatoes in order to classify four Italian cultivation areas (Calabria, Basilicata, Puglia, and Emilia Romagna) and triple concentrated tomato paste samples coming from Italy and from foreign countries (California, China, and Greece). The two alkaline metals (Li and Rb) were the most important variables in the distinction of geographical origin in the discriminating functions. The origin of tomatoes and whether the areas of production of the triple concentrated pastes were inside or outside of Italy were evaluated by three supervised patterns: linear discriminant analysis (LDA), SIMCA, and K-nearest neighbours (KNN). In this case, excellent results were achieved by all models and, in particular, the KNN method correctly classified all samples as originating either from Italy or from outside Italy. Another significant achievement of the proposed method was that the classification was unaffected by the production year for the concentrated paste and the harvesting years for fresh tomatoes.
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Ariyama et al. [17] reported a scientific technique for discriminating onions (Allium cepa L.) collected from Japan (Hokkaido, Hyogo, and Saga), China, United States, New Zealand, Thailand, Australia, and Chile using mineral analysis and LDA. Of 309 samples, 108 were from Hokkaido, 52 were from Saga, 77 were from Hyogo, and 72 were from abroad. The models established by LDA using 14 elements (Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba) were used to discriminate the geographic origin between Hokkaido and abroad, Hyogo and abroad, and Saga and abroad. The discrimination accuracies obtained by cross-validation between Hokkaido and abroad were 100% and 86%, respectively; those between Hyogo and abroad were 100% and 90% accurate, respectively; and those between Saga and abroad were 98% and 90% accurate, respectively. In addition, it was demonstrated that the elemental fingerprint from a specific production area, which can be read in the crops grown therein, did not significantly change with variations in fertilization, crop year, variety, soil type, and production year if appropriate elements were chosen. Japanese and Chinese vegetables such as garlic, bamboo shoots, and eddoe (Colocasia esculenta) were analyzed to discriminate their geographical origins. Kadokura and Ariyama [24] reported the trace element composition of Japanese and Chinese garlic (Allium sativum L.), finding a significant difference in the concentrations of 12 elements (Li, Na, Mg, P, K, Ca, Mn, Fe, Cu, Sr, Mo, and Cd) between the two. The garlic samples were separated according to the amounts of five elements (Li, Na, Mn, Cu, and Zn) by LDA to categorize them into particular groups. All garlic samples (79 samples) were correctly classified in 10-fold cross-validation. Suzuki and Nakashita [18] reported the carbon and oxygen isotope ratios of the bamboo shoots and eddoe; the d13C values of Japanese bamboo shoots and eddoe were relatively lower than those of their Chinese counterparts, while there was no significant difference in d18O between Chinese and Japanese bamboo shoots and eddoe (Figure 3). Moreover, the distribution of d13C and d18O values for Japanese and Chinese bamboo shoots and eddoe overlapped one another. Because China is one of the largest countries in the world, the distribution of stable isotope ratios in China is expected to be quite large. Specifically, northern China is similar to Japan in geological characteristics such as latitude. Therefore, it is very difficult to clearly discriminate the geographical origin of food products from China and Japan.
5 FUTURE PERSPECTIVES IN PDO AUTHENTICATION OF FRUITS AND VEGETABLES The combined use of multiple analytical methods is needed to improve the accuracy of discrimination of the geographical origin of fruits and vegetables. We determined stable isotope ratios and trace element compositions of bulk materials in apples from various cultivation areas in China and Japan to
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FIGURE 3 The d13C and d18O values of Chinese and Japanese bamboo shoots (A), and eddoe (B) [22].
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FIGURE 4 Distributions of the d C and d O values of Chinese, Japanese (Aomori and Nagano) apples (A) and the corresponding dendrogram using the d13C and d18O values and nine elements (Mg, Ca, Mn, Fe, Ni, Cu, Zn, Ga, As, Rb, Sr, Mo, Cd, Cs, Ba, Tl, and Pb) of them (B).
discriminate their geographical origin (Figure 4). We determined stable isotope ratios and trace element compositions of apples from various cultivated areas in China and Japan to discriminate their geographical origin. Of 188 samples, 98 were from Aomori Pref. (Japan), 42 were from Nagano Pref. (Japan), and 48 were from China. The d13C and d18O values of Chinese apples were significantly higher than those of Japanese apples. However, there was no significant difference between Aomori and Nagano Pref. Using the analytical results of d13C and d18O values and 18 elements (Mg, Ca, Mn, Fe, Co, Ni,
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Cu, Zn, Ga, As, Rb, Sr, Mo, Cd, Cs, Ba, Tl, and Pb), apple samples were analyzed by cluster analysis and canonical discriminant analysis to categorize them into specific groups. As a result, the apple samples were divided into three groups: China, Aomori Pref. (Japan), and Nagano Pref. (Japan). Thus, stable isotope and trace element analysis are potentially useful for discriminating the geographical origin of Chinese and Japanese apples [25]. The previous studies of food authenticity introduced in this chapter reported the analysis of raw foods. However, many fruits and vegetables are also processed into juice and jam or are preserved in syrup, frozen, and canned. Such processed products are also targets of mislabelling. Therefore, analytical methods are required for discrimination of food authenticity of processed products as well as fresh fruits and vegetables. However, processed products include seasonings such as salt, sugar, and spices, which contain trace and light elements. Moreover, they are cooked using various food-processing processes such as cutting, boiling, and grilling, factors which can influence the stable isotope ratios and mineral compositions of the processed products. For example, Fung et al. [26] reported the statistical analysis of experimental data on raw versus frozen spinach. There were significant differences at the 1% level between the calcium, chloride, iron, manganese, phosphorus, potassium, and sodium content and a difference at the 5% level between the magnesium content of raw and frozen spinach. The loss of essential elements in commercial freezing of vegetables is probably due to leaching into the blanching and cooling waters. The differences in retention of elements in different frozen vegetables probably depend upon the different chemical forms of the element in the vegetable tissues and the solubility of these compounds in water. The pre-blanching and blanching processes may damage the vegetable cell membranes, which are through to allow free diffusion of ions between the two systems depending upon time of exposure, degree of maceration, or tissue thickness. In one case, boiled bamboo shoots were subjected to multi-trace elemental analysis to discriminate the geographical origin, but no significant difference was found between Japanese and Chinese boiled bamboo shoots because the trace elements were depleted during processing steps such as washing and boiling. On the other hand, carbon, nitrogen, oxygen, and hydrogen are the main elements from which our bodies are composed and are not vulnerable to food-processing operations. Therefore, although pre-treatment for removing seasoning is required, stable isotope analysis is expected to be a potential tool for discrimination of geographical origin for processed fruits and vegetables.
REFERENCES [1] European Commission. Agriculture and rural development. Quality policy. http://ec.europa. eu/agriculture/quality/. [2] Kelly S, Heaton K, Hoogewerff J. Tracing the geographical origin of food: the application of multi-element and multi-isotope analysis. Trends Food Sci Technol 2005;16:555.
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[3] Gonzalvez A, de la Guardia M. Trace-element composition and stable-isotope ratio for discrimination of foods with Protected Designation of Origin. Trends Anal Chem 2009;28:1295. [4] Rapisarda P, Camin F, Fabroni S, Perini M, Torrisi B, Intrigliolo F. Influence of different organic fertilizers on quality parameters and the d15N, d13C, d2H, d34S, and d18O values of orange fruit (Citrus sinensis L. Osbeck). J Agric Food Chem 2010;58:3502. [5] Rapisarda P, Camin F, Faedi W, Paoletti F, Tabilio MR. New markers for the traceability of organic fruit. Acta Horticul 2010;873:173. ˚ kerstro¨m A, Jaakola L, Ba˚ng U, Ja¨derlund A. Effects of latitude-related factors and geo[6] A graphical origin on anthocyanidin concentrations in fruits of Vaccinium myrtillus L. (Bilberries). J Agric Food Chem 2010;58:11939. [7] Rao R, Bencivenni M, La Mura M, Araujo-Burgos T, Corrado G. Molecular characterisation of Vesuvian apricot cultivars: implications for the certification and authentication of protected plant material. J Hortic Sci Biotechnol 2010;85:42. [8] Rummel S, Hoelzl S, Horn P, Rossmann A, Schlicht C. The combination of stable isotope abundance ratios of H, C, N and S with 87Sr/86Sr for geographical origin assignment of orange juices. Food Chem 2010;118:890. [9] Magdas DA, Puscas R. Stable isotopes determination in some Romanian fruit juices. Isotopes Environ Health Stud 2011;47:372. [10] Benabdelkamel H, Di Donna L, Mazzotti F, Naccarato A, Sindona G, Tagarelli A, et al. Authenticity of PGI “Clementine of Calabria” by multielement fingerprint. J Agric Food Chem 2012;60:3717. [11] Matos-Reyes MN, Simonot J, Lo´pez-Salazar O, Cervera ML, de la Guardia M. Authentication of Alicante’s mountain cherries protected designation of origin by their mineral profile. Food Chem. in press. [12] Bateman AS, Kelly SD, Woolfe M. Nitrogen isotope composition of organically and conventionally grown crops. J Agric Food Chem 2007;55:2664. [13] Rogers KM. Nitrogen isotopes as a screening tool to determine the growing regimen of some organic and nonorganic supermarket produce from New Zealand. J Agric Food Chem 2008;56:4078. [14] Mu noz-Falco´n J, Prohens J, Vilanova S, Ribas F, Castroc A, Nueza F. Distinguishing a protected geographical indication vegetable (Almagro eggplant) from closely related varieties with selected morphological traits and molecular markers. J Sci Food Agric 2009;89:320. [15] Anderson KA, Magnuson BA, Tschirgi ML, Smith B. Determining the geographic origin of potatoes with trace metal analysis using statistical and neural network classifiers. J Agric Food Chem 1999;47:1568. [16] Feudo GL, Naccarato A, Sindona G, Tagarelli A. Investigating the origin of tomatoes and triple concentrated tomato pastes through multielement determination by inductively coupled plasma mass Spectrometry and statistical analysis. J Agric Food Chem 2010;58:3801. [17] Ariyama K, Aoyama Y, Mochizuki A, Homura Y, Kadokura M, Yasui A. Determination of the geographic origin of onions between three main production areas in Japan and other countries by mineral composition. J Agric Food Chem 2007;55:347. [18] Suzuki Y, Nakashita R. Approach to the food authenticity problems by using multi-element stable isotope ratio analysis. Kagaku to Seibutsu 2010;4(8):121 [in Japanese]. [19] Kohl DH, Shearer GB, Commones B. Variation of 15N in corn and soil following applications of fertilizer nitrogen. Soil Sci Soc Am J 1973;37:888. [20] Meints VW, Shearer G, Kohl DH, Kurtz LT. A comparison of unenriched vs. 15N enriched fertilizer as a tracer for N fertilizer uptake. Soil Sci 1975;119:421.
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[21] Nakashita R, Suzuki Y. Stable isotope analysis as a potential index for verifying the geographical origin of apple juice. Bunseki Kagaku 2009;5(8):1059 [in Japanese]. [22] Suzuki Y, Nakashita R, Kobe R, Kitai A, Tomiyama S. Tracing the geographical origin of Japanese (Aomori Prefecture) and Chinese apples using stable carbon and oxygen isotope analyses. Nippon Shokuhin Kagaku Kogaku Kaishi 2012;5(9):69 [in Japanese]. [23] Smith RG. Determination of the country of origin of garlic (Allium sativum) using trace metal profiling. J Agric Food Chem 2010;58:3801. [24] Kadokura M, Ariyama K. Development of technique for estimation of geographical origin of Japanese and Chinese garlic using trace element analyses. Food and Agricultural Materials Inspection Center research report. No. 3101; 2007 [in Japanese]. [25] Suzuki Y, Takeuchi J, Nakashita R, Kobe R, Watanabe I. Tracing the geographical origin of Chinese and Japanese apple using stable carbon and oxygen isotope analysis and trace element analysis. In: Abstract of 5th international symposium on recent advances in food analysis, Prague, November 1–4. 2011. [26] Fung AC, Lopez A, Cooler FW. Essential elements in fresh and in frozen spinach and collards. J Food Sci 1978;43:897.