J Foodchem 2005 09 073

Food
Chemistry
Food Chemistry 100 (2007) 504508
www.elsevier.com/locate/foodchem
Organic acids in two Portuguese chestnut

(Castanea sativa Miller) varieties
Barbara Ribeiro a, Joana Rangel a, Patrcia Valentao a, Paula B. Andrade a,
Jose Alberto Pereira b, Hanna Bolke a, Rosa M. Seabra a,*
a
REQUIMTE/Servico de Farmacognosia, Faculdade de Farmacia, Universidade do Porto, R. Anbal Cunha, 164, 4050-047 Porto, Portugal
b
CIMO/ESAB, Quinta de Sta Apolonia, Apartado 1172, 5301-855 Braganca, Portugal
Received 18 July 2005; accepted 20 September 2005
Abstract
The organic acids composition of two dierent chestnut (Castanea sativa Miller) varieties (Judia and Longal) were determined by
HPLC/UV. In order to check the inuence of the processing over these compounds, samples treated in three distinct manners (roasted,
boiled and fried) were also analysed. The results showed that chestnut is characterized by the presence of seven organic acids: oxalic, cisaconitic, citric, ascorbic, malic, quinic and fumaric acids. The organic acid quantitative prole allows the distinction of the two varieties.
Longal variety samples presented higher organic acids contents than those from Judia variety. The contents of the pair malic plus quinic
acids may be useful for the discrimination of the two varieties. Roasting, boiling and frying procedures lead to signicant reduction of
total organic acids contents.
2005 Elsevier Ltd. All rights reserved.
Keywords: Chestnut; Castanea sativa; Judia; Longal; Processing; Organic acids
1. Introduction
Among the 12 world chestnuts species, Castanea sativa
Miller is the most consumed. This species is predominant
in Portugal, where it has a relevant place at the socioeconomic level, reaching an annual fruit production of 20000
tons (Goulao, Valdiviesso, Santana, & Oliveira, 2001).
The best development conditions are found at altitudes
higher than 500 m and winter low temperatures, as in the
Braganca region (Northeast of Portugal) in which
12,500 ha are used for chestnuts cultivation. In fact, this
region gives origin to chestnuts with Denomination dOrigine Protege (DOP), called Castanha da Terra Fria. Cultivation is almost limited to Judia and Longal varieties
because of their higher nutritional qualities and greater
commercial value. The fruits become ripe around Octo*
Corresponding author. Tel.: +351 222078934; fax: +351 222003977.

E-mail address: rseabra@.up.pt (R.M. Seabra).
0308-8146/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.09.073
ber/November and are mainly consumed roasted, boiled

and fried.
From a nutritional point of view, chestnut is considered
as having interesting characteristics. Chestnuts are among
the main sources of starch (up to 70%) but contain low
amounts of protein (24%) and fat (25%) (Vaughan &
Geissler, 1997a). They present several minerals and vitamins and appreciable levels of bre (Vaughan & Geissler,
1997b).
Previous studies on chestnut have concerned the isolation and structural elucidation of a pyrrole alkaloid (Hiermann, Kedwani, Schramm, & Seger, 2002), determination
of sugars (Mguez Bernardez, De la Montana Mguelez,
& Garca Queijeiro, 2004), modication of structure and
digestibility of the starch upon cooking (Pizzoferrato, Rotilio, & Paci, 1999), the eects of roasting on starch, sugars
and fatty acid composition and on chestnut quality
(Kunsch et al., 2001) and the use of cotyledon storage proteins as markers of the genetic diversity within the species
B. Ribeiro et al. / Food Chemistry 100 (2007) 504508
(Alvarez, Munoz-Diez, Martn-Cuevas, Lopez, & Martn,

2003).
The nature and concentration of organic acids are important factors inuencing the organoleptic characteristics of
fruit and vegetables, namely their avour (Vaughan & Geissler, 1997c). The relative amounts and the presence/
absence of each compound have been considered useful in
taxonomic studies (Coppola & Starr, 1986; Silva, Andrade,
Mendes, Seabra, & Ferreira, 2002; Valentao et al., 2005a),
for the determination of percent fruit content in fruit derivatives (Silva et al., 2002) and also to evaluate food processing (Silva et al., 2004a; Valentao et al., 2005b; Wang,
Gonzalez, Gbur, & Aseiage, 1993). Additionally, organic
acids may have a protective role against various diseases
due to their antioxidant activity (Silva et al., 2004b). To
our knowledge, there is no study concerning the organic
acids in chestnut.
The main purposes of this study were to determine the
organic acids composition of two dierent chestnut varieties (Judia and Longal) and to investigate the inuence of
the processing over these compounds. With this aim, chestnut samples collected in Braganca region were subjected to
three dierent procedures (roasting, boiling and frying) and
analysed by HPLC/UV.
505
Samples 9, 10, 11, 12, 25, 26, 27 and 28 were slashed and
boiled in water (300 ml) for 20 min. Samples 13, 14, 15,
16, 29, 30, 31 and 32 were slashed and fried in unused edible oil (renewed for each sample), in a frying-pan (Electric
CO.FR.1060) at 150 C, for 7 min. After processing,
roasted, boiled and fried samples were peeled and stored
in a freezer at 20 C.
All the samples were freeze-dried in a Labconco 4.5
apparatus (Kansas City, MO) before analysis, yielding
20% of dry matter.
2.2. Standards and reagents
The standards (oxalic, cis-aconitic, citric, ascorbic, malic,
quinic, fumaric, ketoglutaric, succinic, shikimic, pyruvic,
malonic and tartaric acids) were from Sigma (St. Louis,
MO, USA) and from Extrasynthese (Genay, France). Methanol and hydrochloric acids were obtained from Merck
(Darmstadt, Germany) and sulphuric acid from Pronalab
(Lisboa, Portugal). The water was treated in a Milli-Q water
purication system (Millipore, Bedford, MA, USA).
2.3. Solid-phase extraction (SPE) columns
The ISOLUTE C18 non-end-capped (NEC) SPE col porosity; 10 g sorbent
umns (50 lm particle size, 60 A
mass/70 ml reservoir volume) were purchased from International Sorbent Technology Ltd. (Mid Glamorgan, UK).
2. Materials and methods

2.1. Samples
Samples from two chestnut varieties (Judia and Longal) were collected from the trees of a chestnut orchard
(Castanea sativa Miller) in the Braganca region (Northeast of Portugal), in November 2004. Two lots of each
variety were separated and immediately transferred to
the laboratory. Each lot was divided into four groups:
three of them were subjected to three distinct processing
methods commonly used for the consumption of chestnut
and the fourth one was used as control (Fig. 1). Each
sample corresponds to a mixture of 15 chestnuts treated
in the same way.
Control samples (samples 1, 2, 3, 4, 17, 18, 19, and 20)
were peeled and frozen at 20 C. Roasted samples (samples 5, 6, 7, 8, 21, 22, 23 and 24) were slashed and roasted
in an electric oven (Balay, Mod. 508) at 200 C, for 40 min.
2.4. Extraction of organic acids

Organic acids extraction was based on a described procedure (Silva et al., 2002). Each chestnut sample (ca. 5 g)
was thoroughly mixed with methanol (5 ml 50 ml), at
40 C. The methanolic extract was ltered and concentrated to dryness under reduced pressure (40 C). The residue of the methanolic extract was redissolved in acid
water (pH 2 with HCl) (ca. 50 ml) and the aqueous solution
was then passed through an ISOLUTE C18 (NEC) column, previously conditioned with 30 ml of methanol and
70 ml of acid water (pH 2 with HCl). The non-polar compounds were retained and the polar ones, such as organic
acids, were eluted with the aqueous solution. This aqueous
extract was evaporated to dryness under reduced pressure
Castanea sativa Miller
var. Judia
var. Longal
13
6 10 14
7 11 15
8 12 16
17 21 25 29
18 22 26 30
19 23 27 31
20 24 28 32
Fig. 1. Schematic representation of chestnut sampling. N: non-treated; R: roasted; B: boiled; F: fried; 132: number of the samples.
506
(40 C), redissolved in 0.01 N sulphuric acid (1 ml) and

20 ll were analysed by HPLC/UV.
A standards mixture was prepared by dissolving the
compounds in 0.01 N sulphuric acid and 20 ll were analysed by HPLC/UV.
100
3
2
4
80
7
5
60
6
2.5. HPLC analysis of organic acids

HPLC analysis was carried out as previously reported
(Silva et al., 2002) with some modications. The separation
of organic acids was performed with an analytical HPLC
unit (Gilson), using an ion exclusion column Nucleogel
Ion 300 OA (300 mm 7.7 mm) column, in conjunction
with a column heating device at 30 C. Elution was carried
out at a solvent ow rate of 0.2 ml/min, isocratically, with
sulphuric acid 0.01 N as the mobile phase. Detection was
performed with an UV detector set at 214 nm.
Organic acids quantication was achieved by the absorbance recorded in the chromatograms relative to external
standards in the standards solution and the peaks were
integrated using a default baseline construction technique.
Oxalic acid and cis-aconitic acid were quantied together
as oxalic acid. Malic acid and quinic acid were quantied
together as malic acid.
40
20
MP
0
0.00
100
10.00
20.00
30.00
40.00
50.00
60.00
70.0
MP
80
60
5
40
2
2.6. Statistical analysis

The evaluation of statistical signicance was determined
by ANOVA followed by Newman-Keuls test. The level of
signicance was set at p < 0.05.
3. Results and discussion
The HPLC/UV analysis of organic acids showed that
both varieties (Judia and Longal) presented a common
qualitative prole, composed by seven compounds: oxalic,
cis-aconitic, citric, ascorbic, malic, quinic and fumaric acids
(Fig. 2(b), Table 1). None of the varieties exhibited ketoglutaric, succinic, shikimic, pyruvic, malonic or tartaric
acids. Another compound, trans-aconitic acid, was also
found in all of the samples, although in vestigial amounts.
However, this compound is considered to be an artefsact,
resulting from the rapid isomerisation of cis-aconitic acid
(Harborne et al., 1999), a phenomenon also observed with
the standards solution (Fig. 2(a)). So, in this work transaconitic acid content was not considered.
The quantication of the identied compounds showed
that Longal variety (samples 1720) had higher organic
acids content than Judia (samples 14): ca. 251 and
205 mg/kg, respectively (Table 1). In both Judia and Longal varieties ascorbic acid was the compound present in the
highest amount, representing ca. 42.6% and 37.0% of total
acids, respectively (Fig. 3). Fumaric acid was the minor
organic acid, corresponding to ca. 0.2% and 0.4% of total
compounds in Judia and Longal varieties, respectively
(Fig. 3). Some dierences were noticed in the quantitative
proles. Judia variety showed a tendency for higher citric
20
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.0
Fig. 2. HPLC/UV chromatogram: (a) standards solution and (b) chestnut

(non-treated sample). Detection at 214 nm. (MP) mobile phase; (1) oxalic
acid; (2) cis-aconitic acid; (3) citric acid; (4) ascorbic acid; (5) malic acid;
(6) quinic acid; (7) fumaric acid; (d) trans-aconitic acid.
acid contents than Longal (ca. 34.8% and 28.4% of total

acids, respectively) (Fig. 3). Additionally, the content of
the pair malic plus quinic acids was signicantly higher in
Longal variety, corresponding to ca. 33.0% of total compounds, while in Judia variety it represented ca. 21.5% of
total acids (Fig. 3). These results suggest that the relative
amounts of the pair malic plus quinic acids may be useful
for the discrimination of these two Portuguese chestnut
varieties.
Roasting, boiling and frying treatments lead to a significant decrease of the total organic acids contents of the two
varieties, with fried chestnuts exhibiting the higher losses of
compounds (ca. 127 and 155 mg/kg in Judia and Longal
varieties, respectively). Thus, heat appears to be the main
cause of modication of the organic acids contents of
chestnut samples, while hydrolytic reactions of organic
acids complexes seem not to interfere. In a general way,
the total acids content decreased mainly due to the reduction of citric and ascorbic acids. Fumaric acid, which was
already a minor compound in both varieties, completely
disappeared or was reduced to very low amounts upon
each treatment (Table 1). The loss of citric and ascorbic
507
Table 1
Organic acids composition of chestnut (mg/kg, dry basis)a
Sample
Variety
Treatment
Compound
Oxalic acid + cis-aconitic acid
Citric acid
Ascorbic acid
Malic acid + quinic acid
Fumaric acid
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Judia
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
Longal
None
None
None
None
Roasted
Roasted
Roasted
Roasted
Boiled
Boiled
Boiled
Boiled
Fried
Fried
Fried
Fried
None
None
None
None
Roasted
Roasted
Roasted
Roasted
Boiled
Boiled
Boiled
Boiled
Fried
Fried
Fried
Fried
1.9
1.8
1.4
1.3
0.9
2.3
3.4
2.6
2.4
1.7
2.6
3.1
4.2
2.2
0.8
0.8
4.6
7.1
1.4
1.5
2.2
4.1
1.7
1.1
2.3
1.7
2.6
1.6
0.9
3.2
0.3
0.5
90.4
57.5
55.4
49.9
23.6
38.5
4.2
48.9
27.0
34.4
15.0
21.4
57.3
24.4
14.4
13.0
107.4
105.4
45.8
39.6
35.8
45.6
27.1
30.3
16.8
32.7
27.2
16.8
34.0
18.3
17.0
21.6
58.4
45.2
164.3
98.7
18.3
11.4
34.7
35.3
9.4
40.4
100.2
47.4
60.0
45.2
7.7
15.8
81.0
114.2
66.7
93.8
48.8
63.7
31.8
25.1
46.4
47.5
28.4
12.8
70.1
23.4
17.2
11.3
51.3
35.9
44.3
41.7
19.6
30.1
36.8
41.1
26.8
33.7
37.3
32.4
47.5
41.8
9.3
11.7
103.4
113.4
61.6
55.0
65.6
77.2
33.8
50.1
36.0
43.8
44.1
39.0
43.5
72.2
23.0
26.1
0.5 (0.0)
0.2 (0.0)
0.7 (0.0)
0.4 (0.0)
Nq
0.1 (0.0)
0.1 (0.0)
Nq
0.1 (0.0)
0.1 (0.0)
Nq
Nq
Nq
1.4 (0.0)
0.7 (0.0)
0.7 (0.0)
0.5 (0.0)
Nq
Nq
Nq
Nq
Nq
0.1 (0.0)
0.1 (0.0)
Nq
0.1 (0.0)
Nq
(0.0)
(0.0)
(0.1)
(0.1)
(0.0)
(0.2)
(0.4)
(0.1)
(0.3)
(0.1)
(0.0)
(0.2)
(0.2)
(0.0)
(0.0)
(0.0)
(2.2)
(0.2)
(0.0)
(0.4)
(0.0)
(0.0)
(0.0)
(0.0)
(0.1)
(0.1)
(0.2)
(0.0)
(0.3)
(0.0)
(0.0)
(0.0)
Total
(0.0)
(0.0)
(0.0)
(0.2)
(1.0)
(0.6)
(0.0)
(0.1)
(0.4)
(0.0)
(0.6)
(1.8)
(0.0)
(0.0)
(0.8)
(0.3)
(2.9)
(3.9)
(0.4)
(1.3)
(0.2)
(1.3)
(0.2)
(0.0)
(0.0)
(0.4)
(0.2)
(0.4)
(0.0)
(0.6)
(1.2)
(0.0)
(0.0)
(0.0)
(0.1)
(0.5)
(3.7)
(3.3)
(6.3)
(0.7)
(1.1)
(0.0)
(8.2)
(6.8)
(0.0)
(0.0)
(0.6)
(0.4)
(5.5)
(8.8)
(4.7)
(3.1)
(4.1)
(0.8)
(0.8)
(0.0)
(0.0)
(5.9)
(0.2)
(0.1)
(0.0)
(2.6)
(2.0)
(2.0)
(0.6)
(1.0)
(2.5)
(1.7)
(0.9)
(16.2)
(3.7)
(0.3)
(0.4)
(0.2)
(0.9)
(2.5)
(0.8)
(1.0)
(1.0)
(0.4)
(2.0)
(0.3)
(6.9)
(0.0)
(3.4)
(0.1)
(0.0)
(0.0)
(3.1)
(0.2)
(1.4)
(2.5)
(0.2)
(2.9)
(0.8)
(2.3)
202.4
159.6
266.1
192.0
62.4
82.3
124.4
128.0
65.6
110.2
155.1
104.3
169.0
68.5
32.2
41.2
297.6
340.8
176.2
190.3
152.3
190.5
94.4
106.6
101.4
125.6
102.2
70.3
148.6
117.1
57.6
59.4
Nq: not quantied.

a
Results are expressed as mean (standard deviation) of four determinations.
Fig. 3. Organic acids prole of Judia and Longal variety chestnuts. (oxa) oxalic acid; (aco) cis-aconitic acid; (cit) citric acid; (asc) ascorbic acid; (mal) malic
acid; (qui) quinic acid; (fum) fumaric acid.
acids seems to indicate that these acids are more sensitive

to the high temperatures used during processing, a behaviour already described (Silva et al., 2004a).
As far as we know, this is the rst report concerning the
organic acids composition of Longal and Judia varieties of
chestnut. The work herein also indicates that roasting, boil-
ing and frying processes aect the chemical composition of

this nut, which may be important when nutritional and
healthy aspects are considered. The results obtained seem
to indicate that organic acid prole is a tool that deserves
to be explored for the discrimination of varieties and also
for evaluating the processing of these fruits.
508
References
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Food

Organic acids in two Portuguese chestnut

Corresponding author. Tel.: +351 222078934; fax: +351 222003977.

ber/November and are mainly consumed roasted, boiled

B. Ribeiro et al. / Food Chemistry 100 (2007) 504508

(Alvarez, Munoz-Diez, Martn-Cuevas, Lopez, & Martn,

2. Materials and methods

2.4. Extraction of organic acids

Castanea sativa Miller

B. Ribeiro et al. / Food Chemistry 100 (2007) 504508

(40 C), redissolved in 0.01 N sulphuric acid (1 ml) and

2.5. HPLC analysis of organic acids

2.6. Statistical analysis

Fig. 2. HPLC/UV chromatogram: (a) standards solution and (b) chestnut

acid contents than Longal (ca. 34.8% and 28.4% of total

B. Ribeiro et al. / Food Chemistry 100 (2007) 504508

Malic acid + quinic acid

Nq: not quantied.

acids seems to indicate that these acids are more sensitive

ing and frying processes aect the chemical composition of

B. Ribeiro et al. / Food Chemistry 100 (2007) 504508

Silva, B. M., Andrade, P. B., Goncalves, A. C., Seabra, R. M., Oliveira,

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