ARTICLE IN PRESS

JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 17 (2004) 63–67
www.elsevier.com/locate/jfca

Original Article

HPLC determination of sugars in varieties of chestnut fruits

from Galicia (Spain)
M. M!ıguez Berna! rdeza, J. De la Montaña Migue! leza,*, J. Garc!ıa Queijeirob
a
Facultad de Ciencias, Area de Nutricion! y Bromatolog!ıa, Campus de Ourense. Universidad de Vigo, As Lagoas sn.
Ourense 32004, Spain
b !
Facultad de Ciencias, Area de Edafolog!ıa y Qu!ımica Agr!ıcola, Campus de Ourense. Universidad de Vigo, As Lagoas sn.
Ourense 32004, Spain
Received 13 June 2002; received in revised form 14 February 2003; accepted 13 April 2003

Abstract

The major components in chestnut (Castanea sativa Mill) are carbohydrates, mainly starch, followed by
sucrose. This disaccharide is one of the most important parameters for the assessment of the commercial
quality of these fruits. In this study we assessed glucose, fructose and sucrose contents in different varieties
of chestnut (C. sativa Mill) using high-performance liquid chromatography (HPLC), by means of a
universal light scattering detector. We dried the samples using freeze-drying and then we extracted the
sugars by means of an ultrasonic bath, using a hydroalcoholic solution at 80% (v/v). We estimated that the
accuracy of this method had a variance of 1.42% for a sucrose content of approximately 22%. We obtained
a recovery rate of sucrose of 86%. We obtained values of sucrose ranging between 6.5% and 19.5%, whilst
glucose and fructose contents were very low, and ranged between 0.00 and 0.27 for glucose and 0.04 and
0.31 for fructose, with a relation near one, which seems to indicate that they are the result of sucrose
hydrolysis.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Varieties of chestnut fruits; Sugars; Sucrose; Glucose; Fructose; Evaporative light scattering

1. Introduction

Carbohydrates are the major nutrients of chestnuts, with sucrose representing one third of total
sugars (Desmaison and Adrian, 1986). This disaccharide is one of the important parameters for
the assessment of the commercial quality of the fruit, since consumers prefer the sweetest fruits.

*Corresponding author.
E-mail address: jmontana@uvigo.es (J. De la Montaña Migue! lez).

0889-1575/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0889-1575(03)00093-0
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! et al. / Journal of Food Composition and Analysis 17 (2004) 63–67

Charro and Barreiro (1957), followed Scales technique (quoted by these authors) in which the
Feehling reagent is used, for carrying out one of the first studies on the nutritional composition of
Galician chestnuts, where they found a sucrose content of 5.7 g/100 g in fresh matter. French
investigators (Bergougnoux et al., 1978) published a study on the nutritional composition of fruits
from different varieties in their country, and found values for this disaccharide ranging from 7.6
to 16.7 g/100 g of dry matter, whilst reducing sugars ranged between 4.5 and 9.6 g/100 g of dry
matter. These authors however do not specify the method of determination used. Bassi and
Marangoni (1984) determined the content of soluble carbohydrates in Italian varieties of chestnut,
using chromatographic determination with antrone and obtained values then from 7.4 to 8.4 g/
100 g of dry matter. Other authors (Senter et al., 1994) studied sugars and nonvolatile acid
contents in fruits from different varieties of Castanea sativa, using gas chromatography, and
obtained values of 9.2 g of sucrose/100 g of dry matter and traces of glucose and fructose.
We should emphasize that the sugar content of chestnuts is subject to several variations,
according to fruit origin (Hadorn and Jungkunz, 1952) storage and the time since their harvest
(Nomura et al., 1995).

2. Materials and methods

Nine chestnut-producing areas were chosen from five municipalities in the Ver!ın-Monterrei
region (Castrelo do Val, Cualedro, Laza, Oimbra y Vilardevos).! Out of the 15 most predominant
varieties (Bermella, Blanca, Boullona, Calva, Casarella, Corronchuda, Das Viñas, Famosa, Foleiro,
Inxerta, Longal, Monfortiña, Soutiña, Touro and Vilamaesa), 80 chestnut trees were selected. The
number of trees of each variety utilized in this work depended on the greater or lesser abundance
of varieties within the municipality.
In the chestnut ripening season, 100 nuts were gathered from each tree and of these, 25 nuts
were taken at random. After peeling, deep dry freezing, grinding and homogenizing, these
chestnuts were used in triplicate batches to measure the sucrose, glucose and fructose content
using the Nomura method (Nomura et al., 1995) but with the following modifications. A smaller
sample quantity and extract was used with two successive ultrasound bath extractions and further
modifications in the proportion of the mobile phase and in the pump flow.

2.1. Sample preparation

Chestnuts were peeled and frozen and then underwent drying for analysis of dry matter. We
extracted sugars when the sample was dry and ground in a grinder and homogenized.

2.2. Extraction of sugars using an ultrasound bath

We weighed 3 g of dry chestnuts and introduced them in a Erlenmeyer flask of 250 ml, adding a
hydroalcoholic solution at 80% (v/v), before putting the top on and introducing it in an
ultrasound bath for half an hour at 60 C. Then it was centrifuged at 2500 rpm for 15 min. The
supernatant, which contains the sugars of this first extraction, was transferred to a volumetric
flask of 50 ml and the residue introduced in a new Erlenmeyer to carry out the second extraction in
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! et al. / Journal of Food Composition and Analysis 17 (2004) 63–67 65

the same conditions as mentioned above. The floating material was again transferred to a flask of
50 ml which contained the previous floating material and this was diluted in an ethanol solution at
exactly 80% (v/v). This solution contained the sugars from the initial chestnut sample and once
filtered—filters of 0.2 mm—and degasified it was utilized for analysis using liquid chromatography.

2.3. Determination of sugars using liquid chromatography

Sugars are determined in the combined extracts using high-performance liquid chromatography
(HPLC) with a universal evaporative light scattering detector. In the mobile phase we used a
solution of acetonitrile and water—in a ratio 80/20—previously filtered and degasified, like the
sample. The column used was of the amino kind (Teknokroma, kromasil 100 NH2 5 mm
25  0.46 cm2), thermostatized at 30 C in order to avoid fluctuations in detector responses.
Working conditions were: flow rate of 1.5 ml/min, detector temperature 130 C and pressure
40 mmHg. The analyses were performed in triplicate batches.
Before the quantitative and qualitative determination of sugars in the sample, we prepared
standard solutions of different sugars: sucrose, glucose and fructose. With those standard
solutions of different sugars we made calibration lines for each one of the sugars, which were later
used for assessing the concentrations corresponding to the different peaks in the chromatograms.

2.4. Statistical methods

For the statistical analysis of our sampling we have used the Windows SPSS 10.0. Comparisons
were carried out at 95% confidence by application of the Anova and Dunnet Test, which
establishes a comparison of means of sucrose content between the chestnut varieties.

3. Results and discussion

3.1. Extraction of sugars

In order to improve the process of extraction we tested different times of extraction and
consecutive extractions; taking into account the results obtained, we decided to do consecutive
extractions of half an hour each one at 60 C.

3.2. Accuracy and precision of the method used

In order to assess the precision of the method, we used three repetitions of the procedure with
frozen chestnuts, which were simultaneously freeze-dried before extracting the sugars. The
floating materials were injected in duplicate, obtaining a variance coefficient of 1.42%, for a mean
content of sucrose of 22%.
For assessing the accuracy of the method, we began with a chestnut sample divided into two-
halves; one of them was divided into three parts, which were frozen at 80 C and freeze-dried.
The other half was also divided into three parts, which were frozen with a known content in
sucrose, before being frozen and freeze-dried under the same conditions mentioned above. These
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! et al. / Journal of Food Composition and Analysis 17 (2004) 63–67

Table 1
Proportion humidity and means (g/100 g dry product) and standard deviations of the sucrose, glucose and fructose
contents
Varieties % Humidity Sucrose (% CV) Glucose Fructose
Bermella 49.0875.85 11.4572.17 0.0870.12 0.0970.14
(18.95%)
Blanca 58.4771.21 8.6372.94 0.2370.39 0.1370.23
(34.10%)
Boullona 54.8573.18 13.3578.77 0.1670.23 0.2270.19
(65.69%)
Calva 54.5872.83 6.5571.00 0.2070.02 0.1170.09
(15.27%)
Casarella 54.85710.96 18.9570.25 0.1670.23 0.2270.31
(1.32%)
Corronchuda 52.9279.59 13.3973.85 0.2570.06 0.3170.15
(28.75%)
Das Viñas 49.9474.36 16.6775.76 0.1370.13 0.1470.16
(34.55%)
Famosa 51.2578.87 9.2873.32 0.2770.18 0.2570.14
(35.77%)
Foleiro 54.1572.48 9.9172.95 0.2670.05 0.2670.13
(29.77%)
Inxerta 58.9073.41 13.0776.52 0.1870.09 0.3070.08
(49.88%)
Longal 54.1371.88 8.9672.34 0.1770.13 0.1970.11
(26.11%)
Monfortiña 48.3774.22 19.4672.47 0.3070.05 0.2770.12
(12.69%)
Soutiña 59.3570.07 11.2270.46 0.0070.00 0.0870.01
(4.10%)
Touro 52.2374.25 11.3375.41 0.2570.22 0.1870.19
(47.75%)
Vilamaesa 48.6472.66 15.9272.93 0.0470.12 0.0470.11
(18.40%)

six samples were injected in duplicate, after the simultaneous extraction of sugars, obtaining a
recovery percentage of sucrose of 86%.
The values obtained for sucrose ranged between 6.5% and 19.5% (Table 1), in spite of the fact
that the main part of our varieties are in the interval 8–15% (middle–low), in accordance with the
values obtained by Lamelas (2000). These results are similar to those obtained by French
investigators (Bergougnoux et al., 1978) for chestnuts from their country, although they are lower
than those obtained by Scharz (1990) and slightly higher than those found by Senter et al. (1994).
The variety Calva showed the lowest content in this disaccharide (Table 1) and Monfortiña and
Casarella were the sweetest varieties, with percentages of sucrose to 19%, although the fruits of
Das Viñas and Vilamaesa also showed percentages higher than 15%.
The values obtained for glucose and fructose were very low (Table 1), as expected, since we
found no references to these sugars as natural components of the chestnut in bibliography. So, we
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! et al. / Journal of Food Composition and Analysis 17 (2004) 63–67 67

may suppose that these components come from the hydrolysis of sucrose, a hypothesis that agrees
with the value for the glucose/fructose ratio, which is near one.
When we analysed the intravarietal homogeneity of the sucrose content, we obtained
coefficients of variation higher than 15% for all varieties, except for Casarella, Monfortiña and
Soutiña (Table 1). These differences may be due to various factors, i.e., different origin of
chestnuts of the same variety, differences in ripening, etc.
Intervarietal comparison of results showed that the sucrose variables were significantly different
and permitted to differentiate nine pairs of varieties (Bermella–Calva; Bermella–Casarella; Calva–
Casarella; Calva–Soutiña; Calva–Vilamaesa; Casarella–Famosa; Casarella–Longal; Casarella–
Famosa; Casarella–Longal, Casarella–Soutiña and Longal–Vilamaesa).

4. Conclusions
The method proposed for assessing sucrose, glucose and fructose makes it possible to obtain a
variance coefficient of 1.42% for a content of sucrose of 22% and a recovery coefficient of
86%.
The fruits of these varieties show sucrose content similar to French chestnuts.
The fruits of Calva showed the lowest content in this disaccharide, whilst Monfortiña and
Casarella were the sweetest fruits. The values for glucose and fructose are very low, with a ratio
near one, which may be due to a partial hydrolysis of sucrose, and so, they are not natural
components of chestnuts.

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