*2.4. Tocopherol Determination*

All high-performance liquid chromatography grade solvents and TOH standards were purchased from Merck, (Darmstadt, Germany) and LGC (Wesel, Germany), respectively. According to DIN EN 12822, HPLC (LC-20 AT; Shimadzu) was used to measure TOH concentrations of the grounded and homogenized nuts. After saponification with potassium hydroxide and extraction with n-hexane, TOH were separated on an Eurospher 100-5 Diol Vertex Plus Column 250 × 4 mm (Knauer, Berlin, Germany) with a mixture of n-hexane/methyl t-butylether (98/2 v/m) as mobile phase at a flow rate of 1.5 mL/min. Isomers of TOH were determined using a fluorescence detector (λex 295 nm, λem 330 nm; RF-10A XL; Shimadzu) and quantified using external standard calibration curves.

#### *2.5. Quantification of Minerals, Trace and Ultra-Trace Elements*

Hazelnut samples were digested with nitric acid in a closed microwave digestion system (Mars 6, CEM, Kamp-Lintfort, Germany) and multi-element quantification was carried out with an ICP-QQQ-MS 8800 mass spectrometer (Agilent, Waldbronn, Germany) [17]. Calcium, magnesium, manganese, iron, copper, zinc, and cadmium were measured on mass, and arsenic and molybdenum were analyzed in the mass-shift mode using oxygen as a reaction gas to eliminate interferences. Rhodium (1 µg/L) was used as internal standard and helium (3 mL/min) as collision gas. For selenium isotope dilution, the analysis was applied as described [18]. The nebulizer gas flow and parameters of lenses, Q1, collision cell, and Q2 were tuned daily for maximum sensitivity (oxide ratio <1.0% (140Ce16O+/140Ce+), double charged ratio <1.5% (140Ce++/140Ce+), background counts <0.1 cps). For quality assurance, the measurement blanks and recalibration check points were determined periodically every 20 samples. For the verification of the applied method, the certified fish muscle reference material ERM-BB422 (Joint Research Centre, European Commission, Geel, Belgium) was successfully analyzed.

#### *2.6. Statistics*

Replicates were measured as indicated in Tables 1–4. The results were expressed as means with standard deviation (SD) or indicated otherwise in Tables 1–4.

#### **3. Results and Discussion**

The aim of the present study was to comprehensively evaluate the nutrient profile of 15 hazelnut varieties cultivated in Germany by analyzing their protein, ash, fiber, and fat content. In addition, the FA distribution was measured and data on TOH, minerals, and trace elements were collected. The present data are in accordance with previously published data of nutrient profiles in hazelnuts. Though, most of these investigations were carried out in hazelnuts from Turkey. Local variances can be explained by differences in soil composition and weather conditions. For the nuts analyzed in the present study, agricultural conditions did not differ as they were grown on the same ground. Thus, the observed alterations in the nutrient profiles of the hazelnuts studied here likely depend on the cultivar. The data presented here indicate also a high nutritional value of the hazelnuts grown in Germany. Thus, harvesting hazelnuts in Germany, even on commercial scale, could be an interesting option for improving nutrient supply.

#### *3.1. Fat, Crude Protein, Dietary Fiber, Moisture and Ash*

Results for the nutritional properties of 15 hazelnut cultivars grown in Germany are shown in Table 1. Fat is the predominant component and the total fat content varied between the 15 cultivars. While the cultivar Red Lambert contained 64.8 g/100 g fat in relation to the fresh weight, the Corabel variety had a fat content of 47.9 g/100 g. This range is comparable with data published previously. Savage and McNeil compared six varieties grown in New Zealand and described a fat content of 54.6 to 63.2% [19]. Later reports on varietal differences in the fat content of hazelnuts in Turkey and other regions revealed similar results [2,20–23]. Ta¸s and Gökmen reported comparable fat contents in a range of 58.1 to 68.9% for hazelnuts harvested in Turkey in 2014 [24], while another group found somewhat lower total fat contents of 53.4 to 63.5% in six hazelnut cultivars grown in Iran in 2010 [25].


**Table 1.** Composition [g/100 g] of 15 hazelnut cultivars grown under identical conditions in Thuringia, Germany.

Data refer to fresh weight; values are expressed as means ± SD (*n* = 2).

Variations were also found for other macronutrients. The crude protein content was the highest in the Corabel variety (22.1 g/100 g), whereas the Cosford variety contained the lowest (10.2 g/100 g). These results are in line with that obtained in other studies, where protein contents of 12 to 22% have been found [2,19,21,23,26]. Amaral et al. investigated 19 cultivars grown in Portugal and reported lower protein contents ranging from 9.3 to 12.7% [20].

Compared to published values, we found higher contents of dietary fiber. The Webb's Prize Cob variety had an outstanding content of 22.2 g/100 g, whereas the Barcelloner Zellernuss variety contained only 13.4 g/100 g. Other studies reported values in the range of 9.5 to 13.2% [19,27,28]. With an average of 16.6% for the hazelnuts studied here, and especially for the varieties Webb's Prize Cob, Hall's Giant (19.7 g/100 g), and Merveille de Bollweiler (19.5 g/100 g), we found remarkably higher dietary fiber contents.

The content of ash, which allows an estimation about the mineral content, was in the range of 1.9 g/100 g (Red Lambert) to 3.2 g/100 g (Gunslebener Zellernuss). Previous reports have shown similar results [2,19]. Locatelli et al. reported slightly lower ash contents in a range of 1.30 to 2.75% [23].

#### *3.2. Fatty Acid Composition*

The tested varieties showed both differences as well as similarities regarding their FA distribution (Table 2). Palmitic acid (C16:0) accounted for only around 5% in all studied hazelnut varieties. Furthermore, proportions of palmitoleic acid (C16:1c9; data not shown), stearic acid (C18:0), and α-linolenic acid (C18:3c9,c12,c15) did not exceed 2.1%. However, there were noticeable differences in the content of the major FA. Oleic acid (C18:1c9) content varied in the range of 65.1 to 81.7%. The variety Tonda di Giffoni had the highest content (81.7%), while the variety Corabel showed the lowest value (65.1%). Next, linoleic acid (C18:3c9,c12) content differed from 10.3% in the Tonda di Giffoni variety to 26.8% in Corabel. The differences in these FA account for a cultivar-specific pattern of MUFA (66.9 to 83.0%) and polyunsaturated FA (PUFA; 10.4 to 27.0%). The total SFA content did not exceed 7.2%. The total n-3 (omega-3) PUFA content was very low for all hazelnuts (<0.2%). The total

n-6 (omega-6) PUFA value varied from 10.3 (Tonda di Giffoni) to 26.8% (Corabel), depending on the cultivar.

The FA distribution of the analyzed hazelnuts were in good agreement with data previously reported [23,25]. Specific characteristics are the slightly lower levels of stearic acid in our varieties, with the highest value of 2.1% for Pauetet, while Locatelli et al. reported contents up to 4.9% [23]. In addition, five varieties (Cosford, Englische Riesen, Webb's Prize Cob, Corabel, and Gunslebener Zellernuss) have oleic acid contents of less than 70%, which are low compared to literature data. This results in an inversely related high content of linoleic acid in these varieties of >20%, which exceeds the values reported in previous publications [20–24,29]. In previous studies, mostly Turkish varieties or nuts cultivated in southern regions were examined. However, an earlier report on the FA distribution of hazelnut cultivars grown in Iran revealed values comparable to ours [25]. In this study, oleic acid content varied from 64.2 to 81.3% and linoleic acid from 10.0 to 21.1%. Bacchetta et al. reported significant differences between two crop years regarding the FA content of 75 hazelnut cultivars from different countries [30]. This indicates that multiple determinants can influence the nutrient composition and especially the proportions of FA.

**Table 2.** Fatty acid composition (% of total FAME <sup>1</sup> ) of 15 hazelnut cultivars grown under identical conditions in Thuringia, Germany.


<sup>1</sup> FAME, fatty acid methyl esters. <sup>2</sup> LA, linoleic acid. <sup>3</sup> ALA, α-linolenic acid. <sup>4</sup> SFA, saturated fatty acids. <sup>5</sup> MUFA, monounsaturated fatty acids. <sup>6</sup> PUFA, polyunsaturated fatty acids.

#### *3.3. Tocopherols*

Table 3 shows that αTOH is the major form of vitamin E in hazelnuts. Only traces of the vitamers β-, γ-, and δTOH were detected with contents of less than 2 mg/100 g. The variety Juningia contained the highest concentration of αTOH (28.9 mg/100 g), while only 9.9 mg/100 g were found in the variety Hall's Giant. Tocopherol levels found here are in accordance with data from the literature. With a mean of 17.5 mg/100 g, the results herein are comparable to data on hazelnuts from Turkey and Portugal [27,31]. Ta¸s and Gökmen reported the differences between two crop years and described notable decreases in the concentration of TOH for the second year for 14 varieties grown in Turkey [24]. However, another group measured TOH contents in hazelnuts grown in Poland and reported 73.90 mg/100 g αTOH for unroasted hazelnuts [32]. A study comparing nutritional values of hazelnuts mainly collected in Turkey determined total TOH contents in the range from 25.8 to 69.8 mg/100 g in hazelnut kernel oils [33]. Environmental, local, and analytical conditions are possible explanations for these remarkable differences.


**Table 3.** Vitamin E content [mg/100 g] of 15 hazelnut cultivars grown under identical conditions in Thuringia, Germany.

Data refer to fresh weight.

#### *3.4. Micronutrients*

Nuts are known as a good source for minerals, which can contribute to a healthy diet [8]. We measured the amount of magnesium, calcium, manganese, iron, copper, zinc, cadmium, selenium, and arsenic in the 15 hazelnut cultivars. There are considerable differences between the examined hazelnuts regarding mineral, trace, and ultra-trace element compositions (Table 4). Magnesium contents ranged from 148 ± 3 mg/100 g (Tonda di Giffoni) to 213 ± 5 mg/100 g (Merveille de Bollweiler). Köksal et al. determined 15 cultivars of hazelnuts grown in Turkey and measured similar magnesium levels of 144 to 224 mg/100 g [21]. Data from other studies confirm these results [27,34]. In addition, the nuts contained high amounts of calcium. Calcium contents ranged from 140 ± 2 (Ennis) to 247 ± 2 mg/100 g (Cosford) and fit well into the picture previously published for hazelnuts grown in Turkey [27,34]. The lowest manganese content was found in Red Lambert (0.682 ± 0.001 mg/100 g), and the highest was determined for Gunslebener Zellernuss (3.92 ± 0.02 mg/100 g). Özdemir et al. reported comparable results with a range from 1.4 to 2.6 mg/100 g for commercial Turkish hazelnuts [34]. Juningia had the lowest level of iron with a content of 2.88 ± 0.07 mg/100 g, while the highest amount was found in Gunslebener Zellernuss with 4.67 ± 0.03 mg/100 g. These values are comparable with data provided in the literature [2,21,34–36]. Contents of copper ranged from 0.764 ± 0.011 to 2.17 ± 0.03 mg/100 g with highest amounts in the variety Corabel and lowest in the variety Ennis and are similar to those reported by others [33]. Corabel contained also the highest levels of zinc (3.93 ± 0.06 mg/100 g), while the variety Juningia contained only 2.12 ± 0.003 mg/100 g; both values are comparable with those reported in the literature [21,36]. Until now, less is known about the content of the trace element molybdenum in hazelnuts. The recommended value for an adequate intake is 65 µg/day [37]. Özkutlu et al. measured between 0.09 to 0.31 mg/kg molybdenum in hazelnuts grown in Turkey [38]. Our hazelnut varieties can contribute to a sufficient intake with contents ranging from 0.109 ± 0.003 mg/100 g (Tonda die Giffoni) to 0.515 ± 0.008 mg/100 g (Merveille de Bollweiler). Levels of selenium are low in the examined samples with highest contents in Tonda di Giffoni and Pauetet with 5.10 ± 0.20 µg/100 g and 6.25 ± 0.51 µg/100 g, respectively. Hazelnuts grown in Turkey showed higher amounts of selenium [27,36], but it is important to note that the soil content for selenium is very low in Germany [39]. Cadmium was only detected at very low amounts in Gunslebener Zellernuss (0.91 ± 0.06 µg/100 g), all other samples were under the limit of quantitation (data not shown). Arsenic levels remained lower than 4 µg/100 g in all varieties, which complies with data for foods of terrestrial origin [40].


**Table 4.**Composition of minerals, trace, and ultra-trace elements of 15 hazelnut cultivars grown under identical conditions in Thuringia, Germany.

 Data refer to fresh weight; values are expressed as means±SD (*n*=3).
