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Jul 31, 2015 - Hazelnuts belonging to different cultivars or cultivated in different geographic areas can be differentiated by their chemical profile;...
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Impact of Roasting on Identification of Hazelnut (Corylus avellana L.) Origin: A Chemometric Approach Monica Locatelli,* Jean Daniel Coïsson, Fabiano Travaglia, Matteo Bordiga, and Marco Arlorio Dipartimento di Scienze del Farmaco and DFB Center, Università degli Studi del Piemonte Orientale “A. Avogadro”, Largo Donegani 2, 28100 Novara, Italy S Supporting Information *

ABSTRACT: Hazelnuts belonging to different cultivars or cultivated in different geographic areas can be differentiated by their chemical profile; however, the roasting process may affect the composition of raw hazelnuts, thus compromising the possibility to identify their origin in processed foods. In this work, we characterized raw and roasted hazelnuts (Tonda Gentile Trilobata, TGT, from Italy and from Chile, Tonda di Giffoni from Italy, and Tombul from Turkey), as well as hazelnuts isolated from commercial products, with the aim to discriminate their cultivar and origin. The chemometric evaluation of selected chemical parameters (proximate composition, fatty acids, total polyphenols, antioxidant activity, and protein fingerprint by SDS-PAGE) permitted us to identify hazelnuts belonging to different cultivars and, concerning TGT samples, their different geographic origin. Also commercial samples containing Piedmontese TGT hazelnuts were correctly assigned to TGT cluster. In conclusion, even if the roasting process modifies the composition of roasted hazelnuts, this preliminary model study suggests that the identification of their origin is still possible. KEYWORDS: hazelnut, roasting, chemometrics, polyphenols, proteins, fatty acids

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INTRODUCTION Roasting is a crucial step in hazelnut processing: beside the positive effects related to food safety (reduction of allergenicity and aflatoxins),1,2 it improves flavor and color of kernels, also leading to a desirable crispy and crunchy texture.3−5 Roasting strongly affects the composition of raw hazelnuts. Several papers have been previously published about this. Alasalvar and coauthors evaluated the impact of roasting on taste-active compounds, such as sugars, organic acids, condensed tannins, and free phenolic acids, evidencing a significant loss in condensed tannins and gallic acid.6 In contrast, the effect of roasting on sugars and organic acids was not noteworthy. In the same manner, roasting had minor influence on hazelnut fatty acid profiles.7 The relative proportions of the individual fatty acids, triacylglycerols, sterols, tocopherols, and tocotrienols were essentially not altered during roasting, while a modest decrease of phytosterols (maximum 14.4%) and vitamin E homologues (maximum 10.0%) was observed.8 Pelvan and coauthors highlighted significant losses in total phenolic content, antioxidant activity, proanthocyanidins, and phenolic acids, suggesting a strong impact due to the skin removal.9 Schmitzer and coauthors reported no significant differences between the antiradical properties of raw and roasted hazelnuts without skin.10 Furthermore, it should be noted that the antioxidant properties of roasted hazelnuts depends on the balance of thermal degradation of natural compounds (such as polyphenols) and the formation of new antioxidant molecules derived from the Maillard reaction (i.e., melanoidins). This is consistent with the evidence that the extension of the roasting times can produce the increase of antioxidant properties,11 even though this effect can be different depending on food matrix, roasting conditions, © XXXX American Chemical Society

extraction and fractionation protocols, and methods employed to evaluate the antioxidant properties. “Tonda Gentile Trilobata” (TGT) hazelnuts from Piedmont, previously known as “Tonda Gentile delle Langhe” (TGL) and covered by PGI designation “Nocciola Piemonte” when cultivated according to the corresponding disciplinary of production, are recognized as high quality hazelnuts with good sensorial characteristics and interesting technological properties. Several approaches have been proposed for the authentication and traceability of these hazelnuts, considering different quality parameters and analytical techniques (ICP− MS, GC×GC-qMS, 1H NMR).12−14 In a previous work, we proposed a chemometrical approach based on both chemotyping and genotyping of unroasted hazelnuts, evidencing that chemotype allows discrimination of TGT hazelnuts cultivated in different countries (Italy and Chile), while RAPD markers are useful to discriminate among cultivars but not to distinguish hazelnuts of the same cultivar from different geographic origins.15 Considering that roasting is a thermal process that strongly influences the chemical composition of hazelnuts and that hazelnuts are generally consumed after roasting, our intent was to identify TGT hazelnuts from Piedmont (Italy) after roasting and in commercial confectionery products (chocolates and pralines), on the basis of their chemical profile. We characterized both raw and roasted hazelnuts (Tonda Gentile Trilobata, TGT, from Italy and from Chile, Tonda di Giffoni from Italy, and Tombul from Turkey), as well as hazelnuts Received: April 1, 2015 Revised: July 27, 2015 Accepted: July 31, 2015

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DOI: 10.1021/acs.jafc.5b03201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Proximate Composition and Fatty Acid Quantificationa sampleb 1TGT 1TGT-160 1TGT-180 2TGT 2TGT-160 2TGT-180 3TGT 3TGT-160 3TGT-180 4TG 4TG-160 4TG-180 5T 5T-160 5T-180 6CL 7CB 8CF 9FR 10CG 11RS a

moisture (%) 3.18 3.05 3.04 3.69 3.13 3.11 3.26 3.12 3.10 4.28 4.06 4.05 3.69 3.67 3.50 2.27 3.37 2.66 3.30 2.54 2.41

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.09 0.01 0.01 0.06 0.01 0.03 0.09 0.03 0.01 0.09 0.19 0.00 0.20 0.14 0.12 0.17 0.08 0.22

proteins (%, dw) 12.1 13.0 13.2 12.9 13.0 13.2 11.8 12.6 12.6 14.0 15.2 14.9 14.2 15.4 15.2 15.0 12.2 12.7 12.8 13.5 17.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.1 0.1 0.2 0.3 0.1 0.2 0.2 0.3 0.2 0.1 0.1 0.3 0.1 0.3 0.1 0.2 0.2 0.2

ashes (%, dw) 2.13 2.23 2.36 2.07 2.13 2.14 2.36 2.42 2.35 2.34 2.69 2.75 2.12 2.54 2.24 2.49 2.33 2.29 2.31 2.25 1.30

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.09 0.06 0.09 0.06 0.08 0.06 0.07 0.01 0.15 0.13 0.02 0.12 0.15 0.13 0.16 0.10 0.13 0.19 0.10 0.04

lipids (%, dw) C16:0 62.7 64.2 64.4 63.3 64.4 64.5 63.5 64.4 63.4 61.3 64.5 62.9 63.6 64.6 64.5 65.5 65.1 67.2 65.4 64.4 67.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 0.7 0.8 1.2 0.8 2.4 0.3 1.0 2.0 0.8 0.9 0.7 0.4 1.4 1.4 0.8 1.3 1.7 0.6 1.4 1.8

6.14 6.48 6.13 6.18 6.15 6.11 6.92 7.16 7.15 5.55 5.45 5.56 5.55 5.54 5.57 7.79 7.67 8.49 7.77 6.60 6.56

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

C18:0 0.04 0.07 0.10 0.10 0.30 0.02 0.01 0.26 0.09 0.02 0.06 0.06 0.15 0.10 0.08 0.16 0.32 0.92 0.13 0.10 0.20

3.40 3.31 3.41 2.88 3.02 2.79 2.98 3.00 3.35 2.85 2.96 3.11 2.80 2.73 2.54 4.86 4.05 4.92 2.88 3.40 3.48

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

C18:1Δ9 0.08 0.10 0.03 0.09 0.18 0.09 0.09 0.07 0.03 0.08 0.05 0.06 0.07 0.18 0.06 0.25 0.06 0.38 0.09 0.10 0.08

81.9 81.4 81.9 83.2 84.9 82.7 80.8 80.2 80.4 80.2 79.6 81.1 80.7 82.0 80.3 76.8 80.5 78.5 77.8 82.0 79.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

C18:1Δ11 0.2 0.2 0.1 0.3 2.7 0.1 0.4 0.4 0.1 1.3 0.4 0.2 0.4 1.2 0.2 0.6 0.4 2.3 0.2 0.2 2.0

1.36 1.46 1.29 1.30 1.31 1.27 1.61 1.65 1.53 1.77 1.11 1.16 1.39 1.15 1.24 1.33 1.43 1.60 1.34 1.34 1.38

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.14 0.01 0.07 0.12 0.01 0.06 0.16 0.03 0.04 0.02 0.09 0.12 0.05 0.08 0.03 0.12 0.43 0.11 0.07 0.11

C18:2 7.23 7.37 7.25 6.49 6.66 7.12 7.73 7.97 7.57 10.90 10.89 9.06 9.61 8.49 10.33 9.24 6.29 6.17 10.05 6.51 8.98

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.15 0.11 0.05 0.15 0.15 0.15 0.26 0.06 0.02 0.22 0.16 0.17 0.20 1.56 0.17 0.21 0.14 0.24 0.22 0.08 2.18

Relative percentage of FAMEs, expressed as mean ± SD (n ≥ 3). bTGT, Tonda Gentile Trilobata; TG, Tonda di Giffoni; T, Turkish. Gentile Trilobata hazelnuts from Piedmont (Italy). Whole hazelnuts were separated by melting chocolate at 40 °C, and any chocolate residue was removed using filter paper. For each commercial product, at least 200 g of hazelnuts was obtained; afterward, the hazelnuts were pooled and subsampled in three portions (each 50 g) that were independently analyzed. Proximate Composition Analysis. The proximate composition was determined as previously described.15 Raw, roasted, and commercial hazelnuts were ground in a mortar (particle size 5T > 3TGT > 1TGT > 4TG (Table 2). After roasting, a strong decrease was observed (from 60% in 4TG-160 to 85% for 2TGT-180). These results are consistent with those reported by Pelvan and coauthors,9 which observed a loss of total phenols after roasting (140 °C for 30 min) from 42% to 88%, but differ in the absolute values, being about 3.5-fold higher. This difference could be ascribed to the different extraction protocol (80% acetone as solvent) but also to the different cultivars analyzed, their geographical origin, and the harvest season. The loss of polyphenols after roasting is prevalently due to removal of hazelnut skin (perisperm), which is an important source of phenolic compounds containing about 40-fold higher polyphenols than kernels.20,26 Furthermore, the high temperatures applied during the process could cause the degradation of thermally sensitive compounds, thus negatively influencing the total phenol content. The hazelnuts isolated from the commercial products showed values in the range or lower than that obtained for the hazelnuts experimentally roasted in laboratory (from 1.59 to 3.57 mg GAE g−1 for 11RS and 9FR samples, respectively). In a general way, commercial samples did not evidence D

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Journal of Agricultural and Food Chemistry Table 3. Flavonoid Content of Hazelnut Samplesa Cat (mg g−1, dw) 1TGT 1TGT-160 1TGT-180 2TGT 2TGT-160 2TGT-180 3TGT 3TGT-160 3TGT-180 4TG 4TG-160 4TG-180 5T 5T-160 5T-180 6CL 7CB 8CF 9FR 10CG 11RS

5.59 20.95 16.42 5.39 11.08 11.29 7.55 12.57 12.01 9.55 8.45 14.95 7.32 9.53 11.61 12.17 15.49 15.62 27.23 22.81 25.94

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.20 1.34 2.26 0.51 1.06 2.03 2.10 2.11 1.56 0.88 1.11 1.37 0.23 0.15 1.58 0.62 2.45 0.59 8.78 2.10 4.40

Epi (mg g−1, dw) 2.20 2.87 2.81 4.49 2.83 2.89 3.58 3.07 3.25 2.13 2.96 2.55 3.02 2.62 2.68 2.29 2.21 2.30 2.35 2.54 2.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Mir (mg g−1, dw)

0.13 0.16 0.15 0.26 0.29 0.18 0.20 0.32 0.32 0.15 0.27 0.18 0.29 0.43 0.25 0.03 0.15 0.04 0.15 0.15 0.07

5.52 7.12 6.95 8.16 7.11 7.42 8.10 6.99 7.13 5.04 5.98 6.31 7.05 6.00 6.82 6.41 6.61 5.97 6.94 8.18 6.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.48 0.38 0.11 0.44 0.40 0.16 0.10 0.68 0.42 0.32 0.31 0.33 0.41 0.24 0.38 0.11 0.52 0.21 0.37 0.60 0.33

Quer (mg g−1, dw) 5.40 8.10 7.70 7.93 7.44 7.55 7.99 7.40 7.43 5.84 6.99 7.80 7.50 5.84 6.47 7.61 6.45 5.86 7.62 7.78 6.90

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.36 0.39 0.38 0.62 0.39 0.27 0.34 0.73 0.44 0.44 0.69 0.05 0.03 0.69 0.09 0.12 0.59 0.22 0.97 0.28 0.42

Kaem (mg g−1, dw) 2.92 3.88 3.75 4.42 4.02 4.01 4.34 3.73 3.82 2.65 3.14 3.33 3.58 3.03 3.50 3.64 3.36 3.08 3.52 3.94 3.54

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.34 0.27 0.06 0.33 0.31 0.09 0.41 0.36 0.16 0.28 0.22 0.17 0.41 0.40 0.08 0.09 0.24 0.21 0.17 0.23 0.13

Mean ± SD, n ≥ 3. Abbreviations: Cat, catechin; Epi, epicatechin; Mir, miricetin; Quer, quercetin; Kaem, kaempferol; TGT, Tonda Gentile Trilobata; TG, Tonda di Giffoni; T, Turkish.

a

Figure 1. Electrophoretic separation (SDS−PAGE) of hazelnut proteins. MW, molecular weight (kDa); M, markers.

significant positive effect of roasting on gallic acid concentration (up to 9-fold higher in roasted than in raw hazelnuts), suggesting that this could be due to degradation of polymerized polyphenols (hydrolyzable tannins), and hydrolysis of other glycosylated flavonoids.10 In contrast, Pelvan and coauthors reported a significant decrease of gallic acid in roasted hazelnuts, calculating an average loss of 52.7% and of 75.5% for its free and bound form, respectively.9 Concerning catechin, this is the first time that an increase after roasting has been observed in hazelnut kernels. Schmitzer and coauthors registered a general decrease of flavan-3-ols, suggesting their thermal degradation. Nevertheless, our results are in agreement with observations in other food matrices, cocoa as an example, in which catechin content increases probably because of epicatechin epimerization.27 In this regard, a negative significant correlation between catechin and epicatechin content in roasted hazelnuts was observed (r = −0.5079; p = 0.0446).

hazelnuts (25.4%), presumably because of its higher total phenol content (3.57 mg GAE g−1). Individual phenolic compounds were tentatively identified and quantified by RP-HPLC/DAD; results, expressed as micrograms per gram of hazelnut (dry weight), are reported in Table 2 (free phenolic acids) and Table 3 (flavonoids). Considering raw hazelnuts, the major compound was catechin (mean value 7.08 μg g−1), followed by quercetin (6.93 μg g−1), and miricetin (6.78 μg g−1); among phenolic acids, protocatechuic acid (5.66 μg g−1) was prevalent. The highest catechin content was observed in 4TG hazelnuts, while the other compounds were generally present in higher concentration in the other cultivars. The effect of the roasting process was different depending on both individual compounds and hazelnut cultivar. In a general way, all the phenolic compounds diminished or remained unvaried during roasting, except gallic acid and catechin, which increased. Previously, Schmitzer and coauthors observed a E

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Figure 2. Two-dimensional representation of PCA (raw and roasted hazelnuts) based on complete data set (panel A), proximate composition and fatty acid profile (panel B), phenolic composition and antioxidant activity (panel C), and relative percentage of SDS-PAGE bands at molecular weight 75, 60, 43, 39, and 10 kDa (panel D). C, hazelnuts from commercial products; T, Turkish hazelnuts; TG, Tonda di Giffoni hazelnuts; TGT, Tonda Gentile Trilobata hazelnuts from Piedmont (Italy); TGTc, Tonda Gentile Trilobata hazelnuts from Chile.

Individual polyphenol content of commercial hazelnuts was in the range of experimentally roasted hazelnuts; the only exception was epicatechin, for which significantly lower values were generally observed in industrially roasted products. Protein Fingerprint. Protein fingerprint was evaluated in nondenaturing conditions by SDS-PAGE; as an example, in Figure 1 is depicted the electrophoretic gel representing the proteins extracted from 5T hazelnuts (both raw and roasted) and commercial samples. The protein migration profiles obtained for different hazelnut samples are comparable and similar to others previously reported in the literature.15,28,29 Based on molecular weights and literature data (http://www.uniprot.org), some bands can be referred to well-known hazelnut allergens. Particularly, the 60 kDa band is attributable to 11S globulin (UniProt Q8W1C2), the 50 kDa band to 7S vicilin (UniProt

Q8S4P9), and the 18 kDa band to Bet v 1 omologue (UniProt Q9SWR4), the 15 kDa band can contain profilin (UniProt Q9AXH5) and oleosin (UniProt Q84T21), and the 13.5 kDa band is ascribable to a protein group including lipid transfer protein (LTP, UniProt Q9ATH2). In order to obtain quantitative data for statistical analyses, the relative percentages of individual bands were calculated; 13 common bands were determined. The major bands identified were those at 13.5, 15, and 60 kDa, with mean relative percentages of 23.4%, 18.0%, and 10.5%, respectively. In a general way, no significant variations were observed between raw and roasted samples, while significant differences were determined depending on hazelnut cultivar and origin. Turkish hazelnuts presented the highest values of the 60 kDa band (mean relative percentage 18.0%), followed in the order by Chilean TGT (10.3%), TG (10.09), TGT (10.0%) and F

DOI: 10.1021/acs.jafc.5b03201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Two-dimensional representation of PCA (roasted hazelnuts) based on proximate composition, fatty acids, total polyphenol content, antioxidant activity, and SDS-PAGE bands at molecular weight 75, 60, 43, 39, and 10 kDa (panel A, PC1 vs PC2; panel B, PC1 vs PC3). C, hazelnuts from commercial products; T, Turkish hazelnuts; TG, Tonda di Giffoni hazelnuts; TGT, Tonda Gentile Trilobata hazelnuts from Piedmont (Italy); TGTc, Tonda Gentile Trilobata hazelnuts from Chile.

activity, individual phenolic compounds identified by HPLC), and protein fingerprint (13 common bands identified by SDSPAGE). Analyzing the individual reduced data sets by PCA, we observed that general composition and fatty acids included useful variables to identify hazelnut origin; thus they were successively combined and processed together. The individual plot of the first two principal components (42.97% and 23.52% of the total variance for PC1 and PC2, respectively) permitted separation of TG and T from TGT hazelnuts, while 10CG and 7CB samples, declared to contain TGT hazelnuts from Piedmont, clustered with TGT (Figure 2B). Considering also PC3 (percentage of variance 16.30%), TG were separated from Turkish hazelnuts and Chilean TGT from Italian ones; the chemical parameters significantly contributing to discriminate samples on PC3 were ashes and relative percentage of C18:1Δ9 (correlation coefficient 0.8251 and −0.4498, respectively). The PCA performed on the variables related to phenolic fraction led to a slightly different result. In fact, the graphical representation of the samples on PC1 and PC2 (total variance 70.61%) shows a less clear clustering based on hazelnut cultivar, but it is possible to note three areas in which raw, laboratory roasted, and commercial hazelnuts are prevalently located. In fact, as previously described, roasting process had a strong impact on phenolic compounds and antioxidant activity and, consequently, the hazelnut clustering was significantly influenced (Figure 2C). Protein fingerprint did not permit the identification of different cultivar or origin, except in the case of Turkish hazelnuts, which presented lower values of PC2. The most important variables for Turkish hazelnut discrimination were the bands at molecular weight 39, 75, 43, and 28.5 kDa (which were significantly correlated to PC2 by a positive correlation) and the bands at 15 and 60 kDa (significant negative correlation). In our previous work on raw hazelnuts, we observed that protein fingerprint is useful to differentiate the hazelnut origin.15 This different result seems to be related to the

commercial (8.0%) hazelnuts. This result is in accord with our previous work on unroasted hazelnuts, in which the band at 58 kDa (presumably including 11S globulin) showed the highest values in Turkish (mean relative percentage 30%) and Chilean TGT (26%) hazelnuts.15 In contrast, 39 kDa band showed the lowest values in Turkish hazelnuts. This band was putatively associated with 11S globulin acidic subunit, primarily because of the molecular weight but also for the strong negative correlation with the 60 kDa band (r = −0.7545, p = 7.75 × 10−5). Other unspecified bands at molecular weight 75, 43, and 10 kDa elicited significant differences among hazelnuts of different origin, while the other bands ascribable to major allergens were not significant for the origin discrimination. Chemometric Evaluation of the Data. The chemometric characterization of hazelnuts was performed using a complex data set, in which all the chemical data were collected (33 variables). The principal component analysis led to nine principal components (PCs) explaining about 90% of total variance (cumulative variance for PC1−PC9 88.99%). The representation of the first two principal components (percentage of variance 19.87% and 17.30%, respectively) did not permit a clear discrimination of hazelnuts based on their different origin and cultivar. Even if three subgroups related to TGT (Italian and Chilean), Turkish and TG, and commercial hazelnuts can be tentatively identified, it is evident that raw hazelnuts are generally separated from their corresponding roasted samples (Figure 2A). The visualization of the third principal component (12.34% of the total variance) was not useful to improve the hazelnut separation. Among commercial hazelnuts, the 10CG sample presents high similarity with the TGT cluster (Figure 2A); this result is in accord with the fact that it contains Piedmontese TGT hazelnuts, as declared on the product label. Afterward, chemical variables were grouped in four reduced data sets: proximate composition (moisture, proteins, ashes, lipids), fatty acids (C16:0, C18:0, C18:1Δ9, C18:1Δ11, and C18:2), phenolic fraction (total polyphenol content, antiradical G

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Journal of Agricultural and Food Chemistry roasting process. In fact, even if the overall protein profile of raw and roasted hazelnuts was almost the same, the bands of roasted hazelnuts were generally less resolved and this could have negatively influenced the possibility to discriminate different hazelnuts. Moreover, in order to improve the protein extraction yield, in this work we employed a different extraction solvent (sample buffer instead of NaCl solution), and this change of the analytical protocol could be partially responsible for having obtained different results. However, considering only the bands for which hazelnut samples resulted significant differences (i.e., bands at molecular weight 75, 60, 43, 39, and 10 kDa), the first two principal components permitted separation of Chilean and Piedmontese TGT hazelnuts and Turkish from TG (Figure 2D). Finally, we decided to omit raw hazelnuts from PCA computations, in order to focus attention on roasted samples and avoid misinterpretation of the results due to the impact of roasting on sensitive parameters. In a general way, hazelnuts can be better separated on the basis of their origin; for example, considering the total data set, Chilean TGT hazelnuts are separated from the Italian TGT. In the case of general composition and fatty acids data set, the exclusion of raw hazelnuts had an uninfluential effect on the capacity to discriminate different samples, because these parameters, expressed on a dry weight basis, are not influenced by roasting. The combination of chemical parameters that permitted us to obtain the best discrimination based on hazelnut cultivar and origin was as follows: general composition, fatty acids, total polyphenol content, antioxidant activity, and SDS-PAGE bands at molecular weight 75, 60, 43, 39, and 10 kDa (overall 16 variables). The two-dimensional representation of PCA (PC1 vs PC2, cumulative variance explained 52.74%) shows the discrimination of TGT hazelnuts from those belonging to the other cultivars; on PC2 also TG are separated from Turkish hazelnuts (Figure 3A). Considering the third principal component (12.27% of the total variance), TGT samples cultivated in Chile are clearly distinguished from Italian ones, gathering in a cluster at higher values of PC3 (Figure 3B). The chemical variable mainly responsible for this clustering is the ash content, being the unique one significantly correlated to the third principal component (r = 0.5464, p = 0.0285). The results of PCA were further processed by an unsupervised clustering method, that is, HCPC (hierarchical clustering on principal components). HCPC is an unsupervised method, which combines principal component analysis, hierarchical clustering, and partitional clustering (specifically k-means). In HCPC, PCA is a preprocessing step that reduces the number of dimensions in parameter space. In a second step, hierarchical clustering is performed on the PCA axes using Ward’s criterion. The number of clusters is chosen on the basis of the increase of inertia. Finally, the clusters obtained from the hierarchical tree cut are used to initialize the k-means algorithm, which consolidates the initial clustering. For this analysis, the minimum number of clusters was set to three, and sample partition was performed by cutting the dendrogram at the higher relative loss of inertia. As reported in Figure 4, the hazelnuts were automatically grouped in three different clusters (the clusters are identified by rectangles). The first cluster includes both TG and Turkish hazelnuts, highlighting major similarities within the same cultivar; the second cluster contains all the TGT hazelnuts and three commercial products (9FR, 7CB, and 10CG samples); in the third cluster are included the remaining commercial hazelnuts, more specifically 11RS, 8CF,

Figure 4. Hierarchical classification of roasted hazelnuts by HCPC analysis.

and 6CL samples. Concerning the TGT cluster, two subgroups can be further identified, in which hazelnuts are separated depending on their geographic origin (Chile or Italy). Moreover, it is interesting to note that hazelnuts isolated from 7CB and 10CG commercial products, declaring to contain TGT hazelnuts from Piedmont, were correctly inserted in the TGT cluster, showing high similarity with Italian hazelnuts. Also 9FR sample was associated with TGT hazelnuts but highlighting higher homology with Chilean samples. In conclusion, the chemometric evaluation of chemical parameters selected within proximate composition, fatty acids profile, polyphenols, antioxidant activity, and protein fingerprint, permitted us to recognize hazelnuts belonging to different cultivars and, considering TGT samples, also their different geographic origin (Chile and Italy). Among the parameters considered, phenolic compounds and antioxidant activity were the most influenced by the hazelnut roasting. The identification of hazelnuts based on their chemical profile is important because it can permit their traceability and, consequently, ensure the quality of their derived products. This is fundamental to certify the quality of high value hazelnuts and, particularly, after the roasting process, which drastically modifies the organoleptic and chemical characteristics of raw hazelnuts, compromising their identification. In this contest, the present study, prevalently based on the identification of Tonda Gentile Trilobata hazelnuts, can be considered as preliminary for the traceability of roasted hazelnuts analyzing their chemical composition. In the future, other research will be necessary to confirm our results, more particularly considering hazelnuts from other geographic origin and cultivars and different harvesting years.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03201. Statistical significance for the proximate composition and the fatty acid relative percentage obtained comparing H

DOI: 10.1021/acs.jafc.5b03201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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hazelnut samples each other (Table 1S), comparing raw, lab-roasted, and commercial hazelnuts (Table 2S), and comparing laboratory-roasted hazelnuts from different origin and cultivar (Table 3S). Statistical significance for the total polyphenols, the antioxidant activity, the phenolic acid content (Table 4S), and the flavonoid content (Table 5S) obtained comparing hazelnut samples each other; for total polyphenols, antioxidant activity, phenolic acid content (Table 6S), and flavonoid content (Table 7S) obtained comparing raw, lab-roasted, and commercial hazelnuts; for total polyphenols, antioxidant activity, phenolic acid content (Table 8S) and the flavonoid content (Table 9S) obtained comparing laboratory-roasted hazelnuts from different origin and cultivar. Screeplot of the PCA performed on the complete data set (Figure 1S). Loading plots of the PCA performed on both raw and roasted hazelnuts using the complete data set (Figure 2S), the proximate composition and fatty acid profile (Figure 3S), the phenolic composition and the antioxidant activity (Figure 4S), and the relative percentage of SDS-PAGE bands at molecular weight 75, 60, 43, 39, and 10 kDa (Figure 5S). Loading plots of the PCA performed on the proximate composition, fatty acids, total polyphenol content, antioxidant activity and SDS-PAGE bands at molecular weight 75, 60, 43, 39, and 10 kDa of roasted hazelnuts (PC1 vs PC2, Figure 6S; PC1 vs PC3, Figure 7S). Contribution of the variables defining PCs obtained from the analysis of proximate composition, fatty acids, total polyphenol content, antioxidant activity and SDSPAGE bands (molecular weight 75, 60, 43, 39, and 10 kDa) of roasted hazelnuts (Table 10S) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0321 375774. Fax: +39 0321 375751. E-mail: [email protected]. Funding

This work was funded by a grant from Regione Piemonte (Ricerca Agricola 2008). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AA, antioxidant activity; GAE, gallic acid equivalent; Gal, gallic acid; Proto, protocatechuic acid; pOH, p-hydroxybenzoic acid; Caff, caffeic acid; Cat, catechin; Epi, epicatechin; Mir, miricetin; Quer, quercetin; Kaem, kaempferol; PCA, principal component analysis; HCPC, hierarchical clustering on principal components

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DOI: 10.1021/acs.jafc.5b03201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b03201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX