Altitude effects on fruit morphology and flour composition of two chestnut cultivars

Scientia Horticulturae 176 (2014) 311–318

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Altitude effects on fruit morphology and flour composition of two chestnut cultivars Annalisa Silvanini a , Chiara Dall’Asta a,∗ , Lucia Morrone b , Martina Cirlini a , Deborah Beghè a , Andrea Fabbri a , Tommaso Ganino a a b

Department of Food Science, University of Parma, Parco Area delle Scienze, 59/a, 43124 Parma, Italy Institute of Biometeorology, National Research Council, Via Gobetti, 101, 40129 Bologna, Italy

a r t i c l e

i n f o

Article history: Received 3 February 2014 Received in revised form 2 July 2014 Accepted 3 July 2014 Keywords: Castanea sativa Mill. Elevation Fruit characteristics Proximate analysis Amino acids Fatty acids

a b s t r a c t Environmental conditions may significantly affect both the chemical composition and the morphological parameters of chestnuts. The aim of this work was the evaluation of altitude effects on flour chemical composition and on fruit morphological parameters of different chestnut cultivars (Luetta e Leccardina), grown at two different altitude levels (700 and 1000 m.a.s.l.). In particular, crude protein, crude fat, amino acid and essential fatty acid content were determined on flours. The two genotypes were also studied from a morphological standpoint, by a biometric and descriptive evaluation of fruits at the different altitudes. Compositional and morphological data were statistically evaluated by ANOVA. The altitude influence on chestnut fruit and flour was ascertained; besides significant differences between the two cultivars, equally significant were the differences within each cultivar when plants grown at different altitudes were compared. © 2014 Published by Elsevier B.V.

1. Introduction The chestnut is present in Italy in all regions within a range of altitude levels, depending on latitude. This condition favored the formation of a rich diversity of cultivars, differing for a number of characters, particularly those concerning the fruit, but also for the traits involved in plant tolerance to biotic and abiotic stress. The fruit of chestnut has been of great importance from the alimentary standpoint through centuries, but in the past century its importance has gradually decreased; however, in recent years an upswing of consumption, of both the fresh and transformed fruit, has been observed. Chestnut fruit is interesting from a nutritional point of view: besides being a good source of starch (>70%), it has a good content of proteins (2–4%), fats (2–5%), and fair amounts of minerals, vitamins and fiber. A great deal of research is carried out on the chemical composition of the chestnut fruit, addressing a number of different aspects: alcaloid content (Hiermann et al., 2002), sugars (Míguez Bernárdez et al., 2004), fatty acid (Borges et al., 2007), polyphenols (Vekiari et al., 2008), modifications of starch structure and digestibility after cooking (Pizzoferrato et al., 1999), heat

∗ Corresponding author. Tel.: +39 0521 905431; fax: +39 0521 905472. E-mail address: [email protected] (C. Dall’Asta). http://dx.doi.org/10.1016/j.scienta.2014.07.008 0304-4238/© 2014 Published by Elsevier B.V.

effects on starch, sugars and fatty acids composition, and on fruit quality (Künsch et al., 2001). The morphological features and quality of the product of a given cultivar depend on two main components, genotype and environment. Each cultivated genotype represents a definite agrarian variety (cultivar, cv.); studies made by Künsch et al. (2001) and by Míguez Bernárdez et al. (2004) indicate that the genetic component determines the chemical composition of the fruit, and in some instances it is possible to discriminate the various cultivars according to chemical values. As concerns the environmental component, few studies are available on the variation of morphological characters with reference to environment variables. Garcea et al. (2005) evaluated shoot growth of some chestnut cultivars at three different altitudes, finding significant differences within the same cultivar. Dinis et al. (2011) found ecophysiological changes in C. sativa cv. Judia grown at different altitudes; in this case, differences were noticed with regard to fruit size, which was smaller at higher elevations, although the extent of the difference depended on the year. No research is available on Castanea concerning changes of fruit or flour chemical composition with changing environment; such studies, however, exist for other plant species, such as sunflower (Sobrino et al., 2003). On this specie it appears that seed lipid composition varies with latitude and altitude. The aim of this research was to evaluate how altitude may influence chemical composition of flours and fruit morphology of two chestnut cultivars (Luetta and Leccardina), grown in North-West

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Italy (Ceno and Taro valleys). In particular, great attention was given to the lipid- and nitrogen-containing fraction, on account of the relevant technological role. Moreover, the antioxidant fraction was also considered, in regard of the protective role toward oxidative degradation played along the flour shelf life. Finally, the volatile profile was also described, since very little is known about this fraction in chestnut flour, so far. 2. Materials and methods 2.1. Study area and cultivar choice The research was carried out in the municipality of Albareto (Parma, Italy); two chestnut groves were selected, the first in Folta of Albareto (700 m.a.s.l.), the second in Breda of Albareto (1000 m.a.s.l.). Two cultivars were chosen: Luetta and Leccardina. These cultivars are widely cultivated in the Valleys of the rivers Ceno and Taro, province of Parma (Bagnaresi et al., 1977; Beghè et al., 2013). Both are early ripening, but their agronomical and carpological features are distinct: Leccardina has a small fruit and is suitable for both fresh consumption and transformation (flour); Luetta produces a medium size fruit, less suited to flour production. Four plants of each cultivar, homogeneous as concerns age, size, exposure and agronomical conditions, were selected in the two groves. 2.2. Fruit morphological characterization The description of the fruits of the two cultivars was made according to the methodology proposed by Bellini et al. (2007) Plant material was sampled in September 2008, 2009 and 2010. On each plant the sampling comprised 20 fruits, and samples were taken from all sides of the trees. The samples were taken from the median zone of fruiting shoots, avoiding misshapen or abnormally small or big fruits (with reference to the average of fruit population). 2.3. Flour monovarietal production In September 2008 and 2009, for each chestnut cultivar, 5 kg of fresh fruits were used for flour production. In the year 2010 it was not possible to produce a sufficient quantity of flour because the damage caused by Dryocosmus kuriphilus Yasumatsu heavily reduced production. Fruit drying, for each cultivar, was carried out in a traditional dry kiln. These buildings, little two-floor cabins (metato), have a square or rectangular plan, are built in local stone, and have a slab stone roof. In the ground floor heating is produced by a fire made with wood and scrap chestnut. In the first floor, homogeneous layers of chestnuts are laid for drying on a rack. During drying, which continues for 30 days, fruits are turned over several times and temperature (40 ◦ C) is daily controlled to keep it constant. The samples were milled used a cereal mill in CRA (Consiglio per la Ricerca e la sperimentazione in Agricoltura) – Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d‘Arda (Piacenza, IT). 2.4. Chemical characterization 2.4.1. Proximate analysis The chemical characterization of the analyzed cultivars was made in stages. Each analysis was repeated three times and data was expressed on dry matter, determined by desiccation in stove at 110 ◦ C for 12 h until constant weight. 2.4.1.1. Extraction and quantification of total carbohydrates. Determination of sugars was performed by the Lane and Eynon

volumetric method (AOAC 930.15); 0.5 g of sample were digested with 10 ml of HCl 6 N for 17 min at 68 ◦ C. Quantitative determination was calculated with Lane and Eynon tables (AOAC 930.15), whose values were corrected with the exact title of Fehling reagent. 2.4.1.2. Extraction and quantification of crude fat. Total fat determination was performed with an acid hydrolysis method followed by extraction with a Soxhlet apparatus for 70 min using diethilic ether as the extraction solvent. The residue obtained was dried for 1 h 30 min at 101 ◦ C ± 2 ◦ C, until constant weight, according to the acid hydrolysis method. 2.4.1.3. Extraction and quantification of total protein. Each sample was analyzed in triplicate for total nitrogen by Kjeldahl method in combination with a copper catalyst using a block digestion system Foss Tecator 2006 Digestor (Höganäs, Sweden) and a Foss 2200 Kjeltec AutoDistillation unit (Foss Tecator). The percentage of nitrogen was transformed into protein content by multiplying the total nitrogen by a conversion factor of 5.30, specific for chestnut fruit (McCarty and Meredith, 1988). 2.4.2. Fatty acid (FA) profile The fatty acid profile of each sample was determined in triplicate by GC/MS analysis after trans-esterification to FA methyl esters (EU 2568/91). The results were reported as relative percentage, calculated on the chromatographic area of each peak and expressed on dry matter content. GC/MS analysis was performed by a Hewlett Packard 5890 separation system (GMI Inc., Minneapolis, USA), equipped with a Hewlett Packard 5971 single quadrupole mass spectrometer with an electronic impact source (GMI Inc., Minneapolis, USA). Chromatographic conditions were the following: the column was a 250 mm × 2.5 mm i.d., 250 nm f.t., Carbowax; the injection volume was 1 ␮l; gradient elution was performed using helium as carrier gas: initial conditions at 80 ◦ C, 0–3 min isothermal step at 80 ◦ C, 3–16 min linear gradient to 210 ◦ C, 16–21 min isothermal step at 210 ◦ C (total analysis time: 21 min); injector temperature, 220 ◦ C, source block temperature, 230 ◦ C. MS detection was performed using a full scan mode from 50 to 500. 2.4.3. Dietary fats and antioxidant activity 2.4.3.1. Dietary fats. According to the results of the fatty acid profile, the quantities were evaluated of saturated fatty acid (SFA), cis-monounsaturated fatty acids (MUFA), cis-polyunsaturated fatty acids (PUFA) and the MUFA/PUFA ratio. 2.4.3.2. DPPH radical scavenging activity. Each flour sample (0.1 g) was added with a 5 ml water:methanol solution (70:30, v/v) and magnetically stirred for 1 h. After centrifugation, the supernatant (1 ml) was diluted with methanol (4 ml). The chestnut extracts (0.2 ml) were then mixed with 2 ml of methanol and 1 ml of methanolic solution containing DPPH radicals (0.2 mM). The mixture was shaken vigorously and left to stand for 60 min in the dark (until stable absorbance values were obtained). The reduction of the DPPH radical was determined by reading the absorbance at 517 nm. The antioxidant capacity was then reported as Trolox Equivalent Antioxidant Capacity (TEAC) (Thaipong et al., 2006). All the data are expressed on dry matter content. 2.4.4. Amino acidic profile Each sample was analyzed in triplicate for the determination of free amino acids. In particular, after Soxhlet defatting, an aliquot of the resulting powder (0.2 g) was extracted by magnetically stirring for 20 min with 5 ml bidistilled water added with 0.75 ml trifluoroacetic acid and 0.05 ml internal standard solution (d,l nor-leucine 50 mM in water). The extract was then centrifuged for 3500 rpm at

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4 ◦ C for 20 min. After filtration, the extract was dried by SpeedVac and the residue was redissolved with 1 ml bidistilled water. The sample was then purified on an Amberlite GC120 column: the strong ion-exchange resin was conditioned with HCl 2 N and washed to neutral pH with bidistilled water, then the sample was applied. After a washing step with bidistilled water, the free amino acids were eluted with 2 N HCl (6 ml) followed by 6 N HCl (2 ml). The eluate was then dried by SpeedVac and the residue was redissolved with 1 ml bidistilled water before HPLC analysis. After a derivatization step according to the AccQ-Tag protocol (Waters, Milford, MA, USA) each sample was analyzed on a C18 AccQ-Tag column (3.9 mm × 150 mm) (Waters, Milford, MA, USA). A gradient elution was performed according to the AccQ-Tag protocol, using a phosphate buffer solution as eluent A and acetonitrile:water 60:40 v/v as eluent B. The temperature was set at 37 ◦ C. The flow rate was 1 ml/min. The fluorescent detector parameters were set as follows: ex = 250 nm, em = 395 nm, gain 1, eufs 100. All the data are expressed on dry matter content. 2.4.5. Volatile profile The volatile fraction of flours was analyzed using solid phase microextraction technique (HS-SPME) coupled with GC/MS. For each SPME analysis, 3 g of flour were placed in a 30 ml glass vial, adding 200 ␮l of a toluene aqueous solution (250 ppm), in accordance with the method utilized by Cirlini et al. (2012). Identification of volatiles was obtained both by comparing mass spectra recorded with library mass spectra (NBS75K, WILEY275) and by Kovats Indices calculation. Since all the compounds considered for this study have been already identified and reported in our previous studies (Cirlini et al., 2012), identification data were not reported in this paper. 2.5. Statistical methods Biometrical and chemical data was statistically elaborated by descriptive and variance analysis (ANOVA), followed by Tukey’s test (p ≤ 0.05) using SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL, 2003). 3. Results 3.1. Fruit morphological characterization The main morphological characteristics considered within this study are reported in Tables 1 and 2. While cv. Luetta is characterized by a large sized fruit, weighing about 12 g, cv. Leccardina has smaller fruit, which weigh less than 10 g. Nut shape is also different between the two cultivars, and the shape index (expressed as ratio among fruit Height, Width and Thickness) is actually higher for cv. Leccardina, which means that its fruit is more elongated that in cv. Luetta. If we consider the year effect, fruit biometrical data show the same trend for almost all characters, i.e., the maximum value occurs in the year 2008, followed by a decrease in 2009 and by a new increase in 2010 (Table 1). Taking into account the biometrical features of the fruit of the two cultivars at 700 and 1000 m.a.s.l., it appears that the Luetta characters remain unchanged, with the exception of fruit thickness which decreases (Table 2). The figures concerning cv. Leccardina, however, indicate an increase of almost all characters at 1000 m.a.s.l., except for the hilum scar, which remains unchanged (Table 2). 3.2. Chemical characterization The effect of the genotype on the chemical composition of chestnut fruits have been reported by several authors so far (Borges et al.,

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2007; Barreira et al., 2009; De Vasconcellos et al., 2009; Neri et al., 2010), while little is known about the effect of the environmental parameters such as altitude. Among the compounds commonly used for these studies, both primary (i.e. starch, proteins, triacylglycerols) and secondary (i.e. organic acids, tannins) metabolites have been taken into consideration. Since this study was focused on flours intended for food production, the efforts were addressed at the investigation of the lipid and nitrogen-containing fractions, due to their technological and nutritional relevance. The wellknown content of MUFA and PUFA in chestnut flour are, indeed, a plus for its technological use as ingredient for the production of “functional food”. Similarly, the chestnut flour is characterized by a gluten-free protein fraction, being thus of great interest for the production of gluten-free products (Dall’Asta et al., 2013; Aponte et al., 2014). In this context, also the amount of antioxidant compounds was investigated, since these substances may exert a dual role in flour: as first a higher antioxidant activity may lead to a longer shelf life, on account of a better prevention of oxidative degradation phenomena; then, a good level of antioxidant compounds is of nutritional interest in the formulation of functional foods. Since the formation and degradation of volatile compounds is strongly related to the environment, the volatile fraction was also investigated within this study. Very few is known so far about this fraction in chestnut flours, in spite of the key role played by organoleptic parameters for the consumers’ acceptance of a food product. 3.2.1. Proximate analysis Data obtained for proximated analysis of the monovarietal chestnut flour considered within this study are reported in Table 3. The chestnut flours of our experiment have moisture values ranging from 4.17% of cv Leccardina from 1000 m.a.s.l., to 5.31% of cv Luetta, again from 1000 m.a.s.l. Concerning starch, data obtained within this study showed an average content of about 80% for the considered cultivars. The lipid fraction of flours (Table 3) is about 4.6%, and it did not show significant differences in the two years. The same applies to comparisons of fats content within the cultivars at different elevations. Protein percentage in the studied flours appears to be influenced by both altitude and cultivar (Table 4). The highest values were found in 2008; and as far as cultivars are concerned a significant increase in protein levels (up to 5.453%) was found in cv. Luetta at the highest altitude (Table 4) 3.2.2. Fatty acid profile The GC/MS analysis of the studied monovarietal flours fatty acids has detected the presence of 5 fatty acids: palmitic (C16), stearic (C18), oleic (C18:1 9), linoleic (C18:2 9,12) and linolenic (C18:3). Oleic acid is present in the highest quantities, with values ranging between 38 and 42% (Table 3) Linoleic acid is the second most represented fatty acid, with 34–38% of the total. The third is palmitic acid, whose values range between 16 and 18%. The least abundant fatty acid is the linolenic (3–6%). The statistical analysis did not show significant differences between the fatty acids profile of 2008 and that of 2009, apart from the levels of linolenic (polyunsaturated) and stearic (saturated) acids. Both undergo a marked reduction in 2009. Minor differences exist in fatty acid composition of flours of 2008 and 2009; this might be due to seasonality, as shown by Borges et al. (2007). The studied cultivars did not show significant differences in their fatty acids profile (Table 4), with the exception of linolenic acid in cv. Luetta, which increased when the fruit was harvested at 1000 m; an increase mirrored by a little increase of the content in polyunsaturated fatty acids (PUFA).

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Table 1 Years effect on fruit characteristics. The data are collected in three vegetative seasons (2008, 2009 and 2010). Year effect

2008

2009

2010

Pr > Diff 2008 vs 2009

Pr > Diff 2008 vs 2010

Pr > Diff 2009 vs 2010

Fruit measured characters Height (cm) Width (cm) Thickness (cm) Width of hilum scar (cm) Height of hilum scar (cm) Weight (g) Weight without pericarp (g) Volume (ml) Shape index

2.771a 3.368a 1.862ab 2.464a 1.232a 11.783a 9.430a 10.725a 0.450b

2.495c 2.964c 1.753b 2.124b 1.183a 8.457c 6.416b 8.018c 0.501a

2.600b 3.169b 1.898a 2.431a 1.340a 10.509b 9.500a 9.388b 0.468ab

<0.0001 <0.0001 0.104 <0.0001 0.896 <0.0001 <0.0001 <0.0001 0.008

<0.0001 <0.0001 0.789 0.878 0.592 0.008 0.981 0.003 0.525

0.010 <0.0001 0.020 <0.0001 0.330 <0.0001 <0.0001 0.003 0.136

The significance of measured fruit characters was evaluated by ANOVA statistical analysis and Tukey test (p ≤ 0.05). Significance is indicated on the rows using different letters. Bold indicates significant variable.

Table 2 Cultivar effect in fruit characteristics at different altitudes. Cultivar effect

Luetta 700 m.a.s.l.

Fruit measured characters

Mean

Height (cm) Width (cm) Thickness (cm) Width of hilum scar (cm) Height of hilum scar (cm) Weight (g) Weight without pericarp (g) Volume (ml) Shape index

2.861a 3.433a 1.999a 1.401a 1.276a 12.613a 10.406a 11.383a 0.438b

Luetta 1000 m.a.s.l.

Leccardina 700 m.a.s.l.

Leccardina 1000 m.a.s.l.

SD

Mean

SD

Mean

SD

Mean

SD

0.312 0.379 0.414 0.487 0.228 3.734 3.378 3.604 0.108

2.742a 3.403a 1.917ab 2.392a 1.236a 11.543a 9.698a 10.417a 0.438b

0.195 0.312 0.340 0.497 0.313 3.224 3.275 2.842 0.099

2.313c 2.755c 1.653c 2.109b 1.297a 7.313c 5.880c 6.891c 0.528a

0.198 0.315 0.287 0.420 1.323 2.283 1.954 2.073 0.114

2.573b 3.076b 1.781bc 2.325a 1.197a 9.529b 7.844b 8.817b 0.488ab

0.287 0.315 0.323 0.392 0.239 2.560 2.315 2.439 0.113

The significance of measured fruit characters was evaluated by ANOVA statistical analysis and Tukey test (p ≤ 0.05). The data were collected in three vegetative seasons (2008, 2009 and 2010). In the table the variability within the cultivar (Luetta and Leccardina) is evaluated for the two altitudes. Significance is indicated on the rows using different letters. SD, standard deviation.

3.2.3. Antioxidant activity The antioxidant activity of monovarietal chestnut flours, mainly due to polyphenols from the epicarp, was determined within this study, as reported in Tables 3 and 4. In general, the antioxidant capacity is higher in 2008, with a value expressed in Trolox mEq of 4.243, while in 2009 it was 2.678 (Table 3); the antioxidant activity of the individual cultivars at 700 and 1000 m.a.s.l. remained unchanged (Table 4). Values obtained for the examined flours are within the average of TEAC obtained by Pellegrini et al. (2003) for other fruits.

3.2.4. Amino acidic profile The amino acidic profile of monovarietal chestnut flours was determined, resulting in the detection of 16 out of 20 proteic amino acids (Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr e Val), as reported in Tables 3 and 4. From our data, no significant differences exist in the two years as to the contents in Ala, Arg, Glu, Leu, Met, Thr, Tyr, e Val; a reduction is detected, from 2008 to 2009, in the content of Asp, His, Ile, Lys, together with an increase in Phe, Pro, Ser (Table 3). With reference to the different elevations, the behavior of the two cultivars is different. Cv. Luetta at 1000 m.a.s.l. shows higher values for Ala, Asp, Glu, Ile, Lys, Met, Phe, Ser e Thr. Only Val was more abundant at the lower elevation (Table 4). Cv. Leccardina at the highest altitude shows higher levels of Asp, Glu, Gly, His, Met, Phe, while Ala, Ser, Thr Tyr e Val are present at the highest concentrations at 700 m.a.s.l. As far as the total amount of essential amino acids is concerned, no significant changes were recorded in the two years (Table 3); with regard to cultivars, there is a significant increase in their content at the higher elevation in cv. Luetta; the same applies to cv. Leccardina, but to a not significant extent (Table 4).

3.2.5. Volatile profile The volatile fingerprint of chestnut flours considered within this study was composed by 26 main peaks, already reported by Cirlini et al. (2012) The main volatile compounds found in chestnut flours were aldehydes (about 75% of the total peak area) followed by alcohols (about 8%), being the former mainly ascribable to the lipid peroxidation and degradation occurring upon chestnut drying and milling. In particular, linear aldehydes are obtained from unsaturated fatty acid peroxidation, while aromatic aldehydes such as benzaldehyde are mainly due to amino acidic degradation (Morini and Maga, 1995). The volatile data collected within this study for the chestnut flours considered herein have been compared by ANOVA analysis, as reported in Table 5. Differences in volatile compounds are mainly related to the cultivar, although several compounds seem to be strongly affected by the altitude. In particular, the amounts of d-limonene, 2-heptanol and benzaldehyde are higher at 700 m for both Leccardina and Luetta samples. As reported in our previous paper (Cirlini et al., 2012), these compounds are already present in fresh fruits and remain stable in flour after processing. Thus, they could be regarded as altitude markers for both cultivars. 4. Discussion The difference between monovarietal flours obtained by two chestnut cultivars grown at different altitudes were addressed by using morphological parameters, proximate analysis and profiles of selected secondary metabolites. Fruit morphological features clearly indicate that the two analyzed chestnut cultivars have different carpological characteristics. Biometrical differences influence fruit growth according to the characteristics of the growing season, and therefore of the climatic conditions.

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Table 3 Years effect on flours chemical compositions. Year effect

2008

2009

Pr > Diff

Proximate analysis (g/100 g) Water content Carbohydrates Fats Proteins

5.663a 79.711a 4.689a 5.133a

3.949b 79.068a 4.668a 4.0124b

<0.0001 0.876 0.946 <0.0001

Fatty acids (g/100 g total fat) Palmitic Stearic Oleic Linoleic Linolenic

17.610a 0.850a 39.561a 36.390a 5.659a

17.747a 0.166b 41.412a 37.687a 3.000b

0.881 0.012 0.252 0.320 0.028

17.913a 41.412a 40.687a 1.018a 2.678b

0.640 0.252 0.265 0.307 <0.0001

Dietary fats (g/100 g total fat) and antioxidant activity (mEq of Trolox d.m.) SFA 18.390a MUFA 39.561a PUFA 42.048a MUFA/PUFA 0.955a Total antioxidant activity 4.243a Amino acids (mg/100 g) Ala Arg Asp Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val  Essential amino acids

4.131a 0.801a 1.828a 1.408a 0.145b 0.579a 0.328a 0.422a 0.056a 0.993a 0.858b 5.671b 5.315b 0.272a 0.341a 0.132a 3.641a

4.656a 0.976a 1.588b 1.223a 0.306a 0.265b 0.267b 0.382a 0.016b 0.9389a 0.998a 7.695a 6.750a 0.284a 0.280a 0.145a 3.297a

0.150 0.584 0.001 0.080 <0.0001 0.005 0.001 0.795 0.011 0.064 0.001 0.022 0.016 0.311 0.120 0.143 0.157

Percent values of flours of the two cultivars (Luetta and Leccardina) at different altitude: Folta (700 m.a.s.l.) and Breda (1000 m.a.s.l.). The data (collected for two years, 2008 and 2009) was analyzed using ANOVA statistical analysis and Tukey test (p ≤ 0.05). Significance is expressed on the rows with different letters and by level of significativity expressed by Pr > Diff. Bold indicates significant variable.

Our data partially agree with the research made by Orˇsanic´ et al. (2009), who studied the influence of altitude on Sorbus torminalis (L.) Crantz fruit size, with the conclusion that the fruit increases its size with increasing elevation; the same authors ascribe this effect to the different climatic and soil conditions existing at the three monitored altitudes. Dinis et al. (2011) observed that the fruit of cv. Judia increase in size with increasing altitude; our data indicate that the response to altitude depends on the cultivar: cv. Luetta fruit do not show appreciable differences at the two altitudes, while cv Leccardina produces lager fruit at 1000 m.s.l.m. (higher elevation). According to the fruit morphology, the Luetta and Leccardina cultivars display different behaviors at the two altitudes; only Leccardina shows significant differences, since the fruit has lower values for all considered characters at 700 m.a.s.l. This might be due to a better adaptation of this cultivar to the colder climate (1000 m.a.s.l.). As far as proximate composition is concerned, the data available in the literature on chestnut flour are poor; however, much more has been written on the chemical composition of fresh or cured fruit. The fresh chestnut has a very high water content, above 50% (Pereira-Lorenzo et al., 2006): this data, together with the high post harvest metabolic activity and the presence of an unlignified pericarp, clearly indicates that the chestnut is a perishable fruit (Sacchetti and Pinnavaia, 2005) The need to preserve this staple food for long periods determined the development of storage methods, such as fruit desiccation and milling into flours. Moisture values found within this study are lower than those reported by Sacchetti et al. (2004) for a cultivar (Castanea sativa

Mill. cv. Pastinese) widespread in the Modena (Italy) province. This is probably due to the traditional dessication process used to obtain the flours considered herein. Chestnuts can be defined as starchy fruits on account of the total sugar content (Erturk et al., 2006); this value is particularly high in the flours used for this study, about 80%, probably on account of genetic variability. This interpretation agrees with Pinnavaia et al. (1993) who likewise report a marked sucrose variability, both between the harvest years and the studied clones. Our data on total carbohydrates in cv. Luetta and cv. Leccardina do not show significant differences between years as well as between elevations. This result is not in agreement with the literature, although it must be pointed out that our data are for total carbohydrate content only. Considering the total fat, data obtained in this research are higher than those reported by literature on flours, i.e., 3.6 ± 1.7% (Sacchetti et al., 2005). Neri et al. (2010) who studied fresh cured fruit of italian chestnut cultivars, found an average value of 4.5%, which is referred to Italian chestnut productions only (Sacchetti and Pinnavaia, 2005). Our data therefore confirm, however, the low total fat level of chestnut flours, especially if compared to the values of almonds and hazelnuts (Borges et al., 2007). Concerning the fatty acids profile obtained for the monovarietal chestnut flours, an interesting content of unsaturated fatty acids should be underlined. In particular, linoleic and linolenic acids belong to the category of essential fatty acids, since they cannot be synthesized by humans, and are precursors of eicosanoids, biologically active molecules. The sum of the three main fatty acids (oleic, linoleic and palmitic) resulted to be close to 90%. This value is higher

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Table 4 Cultivar effect in flours chemical compositions on different altitudes. Cultivar effect

Luetta 700 m.a.s.l.

Luetta 1000 m.a.s.l.

Leccardina 700 m.a.s.l.

Leccardina 1000 m.a.s.l.

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Proximate analysis (g/100 g) Water content Carbohydrates Fats Proteins

5.249a 80.506a 4.737a 4.439b

1.025 7.563 0.476 0.680

5.310a 75.784a 4.552a 5.453a

0.973 8.262 0.436 0.894

4.494ab 83.282a 4.808a 4.281b

0.958 5.414 0.420 0.447

4.170b 75.784a 4.626a 4.116b

1.482 11.851 0.365 1.178

Fatty acids (g/100 g total fat) Palmitic Stearic Oleic Linoleic Linolenic

17.462a 0.664a 40.351a 37.769a 3.755b

2.587 0.577 2.580 1.495 0.881

16.706a 0.624a 38.156a 38.349a 6.171a

0.929 0.793 5.090 2.779 1.040

17.990a 0.444a 42.149a 34.586a 4.935b

1.630 0.622 3.091 3.905 1.503

18.502a 0.494a 40.549a 36.930a 3.522b

2.053 0.441 2.326 2.330 0.886

Dietary fats (g/100 g total fat) and antioxidant activity (mEq of Trolox d.m.) 18.126a 2.417 17.329a SFA 40.351a 2.580 38.156a MUFA PUFA 41.523ab 1.910 44.520a 0.974a 0.090 0.871a MUFA/PUFA 2.881a 0.823 3.318a Total antioxidant capacity

1.608 5.090 4.021 0.193 1.300

18.346a 42.149a 39.521b 1.070a 3.390a

2.044 3.091 1.379 0.115 1.246

18.996a 40.549a 40.452ab 1.006a 3.626a

2.361 2.326 2.326 0.098 0.354

Amino acids (mg/100 g) Ala Arg Asp Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val  Essential amino acids

0.281 1.009 0.174 0.272 0.090 0.141 0.064 0.050 0.057 0.056 0.100 0.445 0.751 0.006 0.082 0.007 0.272

0.447 0.015 0.093 0.070 0.159 0.020 0.033 0.044 0.007 0.041 0.084 3.289 0.223 0.026 0.048 0.011 0.035

4.653ab 1.267a 1.907a 1.953a 0.116b 0.729a 0.285b 0.401a 0.025b 1.053a 1.169a 8.154a 2.923c 0.257b 0.180b 0.051c 3.971a

1.299 0.648 0.250 0.292 0.067 0.466 0.043 0.254 0.033 0.087 0.134 0.774 0.388 0.037 0.057 0.006 0.760

4.128bc 0.355a 1.533b 0.962c 0.155b 0.381ab 0.177c 0.492a 0.009b 0.817c 0.603c 6.126a 6.308b 0.205c 0.348a 0.203a 2.887b

0.299 0.031 0.125 0.128 0.073 0.106 0.024 0.518 0.012 0.040 0.057 1.527 0.378 0.005 0.108 0.033 0.510

4.653ab 1.534a 2.106a 1.391b 0.199b 0.447ab 0.406a 0.349a 0.085a 1.061a 1.084a 7.150a 10.510a 0.303ab 0.411a 0.157b 3.891a

6.048a 0.398a 1.287c 0.955c 0.432a 0.131b 0.325b 0.367a 0.026b 0.933b 0.857b 5.301a 4.389bc 0.346a 0.304ab 0.142b 3.120ab

Percent values of flours of the two cultivars (Luetta and Leccardina) at different locations: Folta (700 m.a.s.l.) and Breda (1000 m.a.s.l.). The data (collected for two years, 2008 and 2009) was analyzed using ANOVA statistical analysis and Tukey test (p ≤ 0.05). Significance is expressed on the rows with different letters. SD, standard deviation. Table 5 Volatile compounds found in chestnut flour on different altitudes for the two cultivars (Luetta and Leccardina) at different locations: Folta (700 m.a.s.l.) and Breda (1000 m.a.s.l.). Data are expressed as relative percentage (%). Luetta 700 m.a.s.l. Mean Hexanal d-Limonene 2-Pentylfuran 1-Pentanol Octanal 2-Heptenal 1-Hexanol nonanal 3-Octen-2-one cis-Linalool oxide 2-Octenal 1-Octen-3-ol 1-Heptanol Furfural 4-Ethyl-cyclohexanol 3,5-Octadien-2-one Benzaldehyde 2-Nonenal 1-Octanol 5-Methylfurfural ␥-Butyrolactone 2-Phenylacetaldehyde Acetophenone 5-Furfuryl alcohol 2,4-Nonadienal ␥-Valerolactone

b

49.47 4.30b 0.88bc 0.13b 4.29b 2.17a 2.61ab 7.58a 0.05a 0.15b 4.69ab 0.85b 0.93a 0.89b 3.28a 3.61a 0.39b 0.92a 1.12 a 0.12 a 2.29ab 1.06ab 1.73a 0.80a 0.06b 1.30b

SD 0.51 0.24 0.14 0.01 0.07 0.07 0.44 0.60 0.02 0.01 0.25 0.05 0.04 0.24 0.81 0.05 0.02 0.07 0.15 0.01 0.02 0.05 0.08 0.01 0.03 0.02

Luetta 1000 m.a.s.l.

Leccardina 700 m.a.s.l.

Leccardina 1000 m.a.s.l.

Mean

Mean

Mean

a

52.50 3.53c 0.50c 0.08b 7.56a 1.07bc 3.07a 9.52a 0.36a 0.84a 6.15a 2.00a 0.96a 1.38ab 1.77ab 1.62b 0.08c 0.66b 1.44a 0.01a 1.63b 0.83c 0.52b 0.12c 0.21a 0.42b

SD 0.44 0.01 0.28 0.04 0.71 0.06 0.27 1.57 0.14 0.14 1.09 0.34 0.32 0.17 0.09 0.20 0.10 0.02 0.37 0.00 0.21 0.04 0.13 0.11 0.05 0.35

ab

50.77 5.22a 2.61a 0.32a 6.71ab 1.38b 2.70ab 9.70a 0.09a 0.39b 1.91c 1.11ab 1.17a 0.83b 0.81b 1.81b 2.30a 0.54b 1.52a 0.32a 2.07ab 1.55a 0.27bc 0.31bc 0.32a 0.84a

SD 0.93 0.04 0.07 0.01 0.98 0.03 0.47 0.20 0.01 0.01 0.18 0.31 0.06 0.07 0.02 0.37 0.00 0.01 0.09 0.27 0.01 0.47 0.19 0.02 0.01 0.15

b

48.30 3.21c 1.55ab 0.14b 6.67ab 0.80c 1.44b 10.47a 0.40a 0.43b 3.81bc 1.79ab 1.48a 2.00a 2.32ab 2.89a 0.40b 0.65b 1.46a 0.38a 2.63a 0.90c 1.20ab 0.75ab 0.23a 1.16b

SD 0.65 0.15 0.21 0.03 0.18 0.20 0.13 0.50 0.11 0.00 0.08 0.07 0.12 0.08 0.10 0.02 0.06 0.01 0.19 0.07 0.29 0.07 0.24 0.20 0.01 0.07

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than that (85%) measured by Borges et al. (2007), but in agreement with the data reported by Barreira et al. (2009). It must be noted, however, that both quoted studies were conducted on fresh nuts and not on flours. As from the statistical analysis, linolenic acid, whose content in 2009 is reduced by about 50% if compared to the previous year, is therefore higher in the year 2008, when the average values of minimum temperatures are lower than those recorded in the following year. A similar result was obtained by Wolf et al. (1982) in a research on the effects of temperature on the composition of soy beans: the plants grown at the lowest temperatures had a far higher content in linolenic acid. The data referring to the fatty acids of the studied cultivars can provide important information from the nutritional standpoint, such as the monounsaturated/polyunsaturated fatty acids ratio (MUFA/PUFA). A ␻6/␻3 ratio close to 1 is indeed beneficial, on account of the possible preventive action against cardiovascular diseases and other degenerative disorders (Simopoulos, 2007). This ratio slightly increased in 2009, if compared to 2008, although not significantly; no differences were instead detected in both cvs for the MUFA/PUFA ratio at the two elevations. Due to the high amount of unsaturated fatty acids, chestnut flours can be considered as a substrate prone to oxidative degradation during shelflife. However, the good content of ellagitannins from fruits epicarp can act as a protective factor against peroxidation. In addition, a good antioxidant capacity represents an interesting feature from the nutritional point of view. The antioxidant activity of chestnuts is mainly due to the presence of tannins, mainly located in the episperm. Several research works (Barreira et al., 2008) have measured the total antioxidant capacity of this tissue, demonstrating its extremely high antioxidant activity. In Neri et al. (2010) the antioxidant capacity of chestnut samples was measured on fresh tissue. However, the authors found values between 2.03 and 3.11 mEq of Trolox, in agreement with our results. Among amino acids detected in the considered flours, cysteine was found at lower amount than that reported by De Vasconcellos et al. (2009). It should be noticed though, that those results have been obtained from fresh fruit, while ours were gathered from flours, which have undergone a technological process of desiccation and grinding. Cysteine could be actually degraded by thermal treatment, especially on account of the long desiccation step. In both years the quantitatively most important amino acids were proline, serine and alanine; this result does not agree with the studies of the above mentioned authors, as De Vasconcellos et al. (2009) indicates as main amino acids asparagine (Asp) and glutamic acid (Glu). With reference to the different elevations, a research carried out on winter rye by Griffith et al. (1997) proved the existence of seven “antifreeze” proteins, rich in Asp, Glu, Ser, Thr, Gly and Ala. Four of these amino acids (Asp, Glu, Ser, Thr) are present at high concentrations in Luetta flour at the higher elevation, while in Leccardina cv. only Asp and Glu content increases at the higher elevation. In addition, the data concerning the protein content of flours indicate a significantly higher protein content (about 5.5%) in cv. Luetta at 1000 m.a.s.l. if compared to the levels found in the same cv at 700 m and in cv. Leccardina at both elevations. This higher protein amount might be explained by a lower adaptability of cv. Luetta to a cold environment, with the consequent elicitation of the “antifreeze” protein synthesis as response to the abiotic stress. The larger size of Leccardina fruit at 1000 m.a.s.l. may also be taken as a further confirmation of this assumption. The data discussed above showed, thus, that the flours obtained from the two cultivars are different for some chemical parameters. Cv. Luetta undergoes an increase of the percentage of total proteins when grown at the higher altitude, and an increase of some amino

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acids (Asp, Glu, Ser, Thr) which might be responsible for the synthesis of “antifreeze” proteins, (Griffith et al., 1997). The adaptation to a higher altitude, and therefore to a colder microclimate, can be indicated in both cultivars also by a higher concentration of PUFA, which might be responsible of an increased cell membrane fluidity, needed to cope with an average temperature which is a few degrees lower than that experimented at 700 m.a.s.l. Finally, the analysis of the volatile profiles of different flours showed that d-limonene, 2-heptanol and benzaldehyde could be regarded as altitude markers for both cultivars. It has been reported that terpene emissions in plants are influenced by biotic and abiotic factors, such as light and temperature, obtaining the maximum level in warmer periods (Llusia et al., 2013 and references herein). Accordingly, the highest amount of d-limonene found in chestnut samples harvested at 700 m can be explained on account of the milder climate experienced by trees compared to those grown at 1000 m. Benzaldehyde and 2-heptanol are volatiles deriving from the degradation of the lignin fraction and from the lipid peroxidation, respectively. Both compounds, thus, tend to accumulate along fruit ripening. Accordingly, the lowest levels found in samples collected at 1000 m can be explained considering a 10 days shift in the phenological ripening at that elevation. In general, however, the volatile profiles of chestnut flour seem to be more affected by the genetic background than by the altitude, being the profile obtained for cv. Leccardina at different altitudes more stable than that reported for cv. Luetta.

5. Conclusion The results of this research allow us to draw a few conclusions about the effects exerted by the altitude on the two considered chestnut cultivars. From the phenological point of view (data not reported) a delay of about 10 days can be noticed at higher altitudes for all the phenological stages in both cultivars (full bloom time, end of fruit growth and ripening). As pointed out by our data, different years determine different morphological fruit characteristics, which are unrelated to differences between cultivars in the same year (data not shown). In 2009 there is a reduction in the values of almost all the measured characters, and this might be ascribed to the lower rainfall of that year (source: ISTAT data, 2010). Finally, it appears that the differences recorded at different altitudes are markedly dependent on the cultivar; in our study cv. Leccardina is undoubtedly the best adapted to the higher altitude, concerning both fruit morphology and chemical composition of the flour.

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