Food Research International 44 (2011) 1419–1426

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Solid-state fermentation of red raspberry (Rubus ideaus L.) and arbutus berry
(Arbutus unedo, L.) and characterization of their distillates
Elisa Alonso González a, Ana Torrado Agrasar a, Lorenzo M. Pastrana Castro a,
Ignacio Orriols Fernández b, Nelson Pérez Guerra a,⁎
a
Departamento de Química Analítica y Alimentaria. Facultad de Ciencias, Universidad de Vigo, Campus de Ourense, As Lagoas s/n, 32004, Ourense, Spain
b
Estación de Viticultura y Enología de Galicia. Ponte San Clodio s/n, 32427 Leiro, Ourense, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to obtain two distilled alcoholic beverages from red raspberry and arbutus
Received 29 November 2010 berry by solid-state fermentation and subsequent distillation of the fermented fruits. The mean
Accepted 16 February 2011 concentrations of ethanol and volatile substances in the distillates from red raspberry (41.3 and 200.1 g/hL
aa) and arbutus berry (44.3 and 267.1 g/hL aa) were higher than the corresponding minimum limits (38.5 and
Keywords: 200 g/hL aa) fixed by the European Council (Regulation 110/2008) for fruit distillates. In addition, the mean
Alcoholic beverages arbutus berry
concentrations of methanol in the two alcoholic beverages (113.9 g/hL aa in case of red raspberry, and
Red raspberry
Ethanol
320.5 g/hL aa in case of arbutus berry) were much lower than the maximum levels of acceptability that the
Solid-state fermentation aforementioned regulation fixed for red raspberry (1200 g/hL aa) and arbutus berry (1000 g/hL aa) distillates.
Volatile compounds These results showed that both fruits can be used as fermentation substrates for producing two alcoholic
beverages with high quality, which are safe for the consumers without any repercussion to their health from
methanol concentrations.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Mountain regions of Galicia, an autonomous community located in
north-west Spain, are still largely dependent on subsistence agriculture,
which is essentially focused towards beef cattle and poultry farming.
Nowadays, this region has been characterized by a decreasing
population and diminished income levels in comparison to those of
the urban areas (Alonso, Torrado, Pastrana, Orriols, & Pérez-Guerra
2010; Crecente, Alvarez, & Fra 2002). This indicated the need of creating
infrastructures and facilities that allow an integral management of the
forest and the use of all their resources (Alonso et al., 2010).
The successful production of distilled alcoholic beverages from two
fruits of the forest (black mulberry and black currant), by using a
reproducible fermentation procedure, has been recently proposed as an
alternative that could have a beneficial effect on the economy of the
region (Alonso et al., 2010). Thus, the production of other two distillates
from arbutus berries and raspberries could also be advantageous for the
Galician farmers, because they could offer a wider range of high added
Abbreviations: Aa, absolute alcohol; ABD, arbutus berry distillate; Al, Albariño; Bag,
bagaceiras; BBD, blackberry distillate; BCD, black currant distillate; BMD, black
mulberry distillate; Gd, Godello; Me, Mencia; PEt, ethanol productivity; RBD, red
raspberry distillate; RI, refractive index; Tr, Treixadura; YEt/RSc, ethanol yield from
reducing sugars consumed; WD, whey distillate.
⁎ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.
E-mail address: nelsonpg@uvigo.es (N.P. Guerra).
value products (e.g. distillates from fruits of the forest) and conse-
quently, their incomes could be increased.
Arbutus berries, a good source of antioxidants such as flavonoids,
vitamins C and E and carotenoids, are commonly consumed as
processed products including preserves, jams, marmalades and jellies
(Pallauf, Rivas, del Castillo, Cano, & de Pascual 2008). In addition, these
fruits have been used to treat arterial hypertension (Cavaco, Longuinho,
Quintas, & Saraiva 2007) or as antiseptics, diuretics and laxatives in
traditional folk medicine (Ayaz, Kucukislamoglu, & Reunanen 2000;
Pabuçcuoglu, Kivçak, Bas, & Mert 2003). In the same way, raspberries
(Rubus idaeus L.) have reported to contain high levels of ellagic acid
(Juranic et al., 2005), which has shown to have antiviral activity
(Corthout, Peiters, Claeys, Vanden Berghe, & Vleitinck 1991) and
anticancer potential (Stoner & Morse 1997).
With regard to the production of alcoholic beverages from both
fruits, there are some important problems that interfere with the
reproduction of the fermentation process. On the one hand, with
regard to red raspberry, there is so far little data in the literature on
the production of alcoholic beverages with this fruit (Duarte, Dragone,
et al., 2010). On the other hand, although an aromatic distillate from
arbutus berry (Aguardente de medronho) has been traditionally
produced in the Algarve's region (Portugal) on a small scale (Alarcão-
E-Silva, Leitão, Azinheira, & Leitão 2001), the fermentation process
has not been studied in detail (Cavaco et al., 2007). According to these
researchers, the fermentation is artisanally carried out under
uncontrolled conditions, by the wild microbiota of the fruits during

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.02.032
1420 E.A. González et al. / Food Research International 44 (2011) 1419–1426
4–5 weeks, depending on the weather conditions. In these conditions,
the fruits are over-fermented or not completely fermented leading to
the appearance of some organoleptic defects such as acidity, lack of
flavor, or even off-flavors in the fermented products (Cavaco et al.,
2007). As a consequence, the final quality of the alcoholic beverages
obtained could be highly variable (Aloys & Angeline 2009). An
alternative to solve this problem could be the design of a reproducible
and productive fermentation process in order to obtain alcoholic
beverages with a more uniform quality, as it was proposed before for
black mulberry and black currant (Alonso et al., 2010).
On the other hand, the current commercialization of other known
alcoholic beverages obtained from different fruits such as grapes
(Diéguez, de la Peña, & Gómez 2005; Silva & Malcata 1998; 1999),
mulberries (Darias, Lobo, Hernández, Díaz, & Díaz 2003; Soufleros,
Mygdalia, & Natskoulis 2004), pears (García, Achaerandio, Ferrando,
Güell, & López 2007) and apples (Xu, Fan, & Qian 2007) could facilitate
the market penetration of the two distillates from red raspberry and
arbutus berry.
Therefore, the main goal of this work was to study the fermentation
processes of red raspberry and arbutus berry fruits in order to obtain in a
reproducible way, two distillates with a well-defined final quality, with
methanol concentrations lower than the maximum limit of acceptability
fixed by the European Council (Regulation EEC110/2008) for fruit
spirits. The results of this study will facilitate the improvement and
reproduction of the manufacture process of two new valuable products.
This approach could contribute to the development of new infrastruc-
tures that allow a more integral management of the forest in the Galician
autonomous community as well as an increase in the farmer income.
2. Materials and methods
2.1. Yeast strain
Saccharomyces cerevisiae IFI83, a high ethanol-producing strain,
was obtained from the yeast collection of the Institute for Industrial
Fermentations (IFI), Spanish National Research Council (CSIC),
Madrid, Spain. The yeast strain was grown in a conventional medium
composed by (g/L): bacteriological peptone, 20; yeast extract, 15;
glucose, 20; pH, 6.2. Working cultures were maintained as slants at
4 °C on a medium composed by (g/L): malt extract, 20; yeast extract,
1; agar, 20; pH, 7.2. These cultures were propagated twice in the same
medium at 18 °C before their use as inoculum.
2.2. Fermentation substrates
The fruits used in this paper were red raspberry (Rubus ideaus L.) and
arbutus berry (Arbutus unedo, L.), which were picked at the stage of full
ripeness in different plantations of the Galician Region. Fruits were
manually selected and transported to the Department of Food Analytical
Chemistry and Food Science of the University of Vigo on the same day of
recollection. The fruits were immediately frozen and stored at −40 °C
until further use.
2.3. Physicochemical characterization of the fruits
Frozen fruit samples were thawed at room temperature and crushed
for 5 min in an Ultraturrax at 9500 rpm. Then, 1 g of pulp was poured
into a 100 mL Erlenmeyer flask and mixed with 50 mL of distilled water.
After mixing, the samples were centrifuged (15,000 ×g/5 min) and the
supernatants were used to determine pH and reducing sugars (3.5-
Dinitrosalicylic acid reaction (Bernfeld 1951) with glucose (Panreac,
Barcelona, Spain) as standard).
Total protein (N × 6.25), solid residue, moisture content and ashes
were determined in the undiluted pulp according to the methods
described in a previous work (Alonso et al., 2010).
All of the analyses were done in triplicate and the results were
expressed as means with their respective standard deviations. The
mean composition (wet basis) of the pulps obtained from red
raspberry and arbutus berry fruits are shown in Table 1.
2.4. Solid-state fermentations of the fruit pulps
The fruits were slightly crushed with a mortar and pestle to break all
the berries. Fermentations were conducted in 150 mL Erlenmeyer flasks
previously sterilized, containing exactly 50 g of fruit, and covered with
cotton plugs. In all cases, the crushed fruits used as fermentation
substrates were supplemented with 0.5 mL of a concentrated salts
solution composed of NH4Cl and KH2PO4 to get nitrogen and
phosphorus supplements of 200 mg and 136 mg per kg of fresh pulp,
respectively (Alonso et al., 2010). The moisture contents after salts
supplementation were 90.9% (in red raspberry) and 72.3% (in arbutus
berry). Then, the pulps were fermented by using three fermentation
strategies. In the first, the crushed fruits were spontaneously fermented
with their own indigenous microbiota. In the second and third
fermentations, the thermally treated (105 °C/20 min) and the non-
thermally treated fruit pulps were respectively used as the fermentation
substrates, which were inoculated with a 24-h culture of S. cerevisiae
IFI83.
Inoculation was made by addition of 0.4 mL of a cell suspension of
S. cerevisiae IFI83 adjusted previously to give an initial concentration
of 5 × 105 cells/g of pulp. In the case of the spontaneous fermentation,
the cell suspension was substituted by sterile water. The resulting
moisture concentrations after inoculation were 91.7% in red raspberry
and 73.1% in arbutus berry. The contents of the flasks were mixed
thoroughly and then the cultures were incubated under static
conditions at 18 °C.
Samples as whole flasks in triplicate were withdrawn at regular
intervals for analytical determinations. These fermentation samples
were mixed with 100 mL of distilled water in an Ultraturrax and then
centrifuged (15,000 × g/5 min). The supernatants were used to
measure culture pH and the concentrations of reducing sugars
(Bernfeld, 1951) and fermentation products (ethanol, glycerol and
acetic acid). The concentration (in wet basis) of reducing sugars and
fermentation products were expressed in g/100 g of fruit pulp.
2.5. Fermentation products analysis
Concentrations of fermentation products were quantified by ion
exclusion chromatography using a ICSep ICE-ION-300 TRANSGENOMIC
column with a pre-GC-801 Guard ICSep (mobile phase, 8.5 mM H2SO4;
flow rate, 0.4 mL/min; temperature, 30 °C; RI detection). Solutions of
ethanol, glycerol and acetic acid at a concentration between 1 and 10 g/L
were used as patterns (Alonso et al., 2010).
2.6. Distillation
Fermented pulps were distilled in duplicate by using a steam drag
distillation system equipped with a distilling flask fixed to a rectifying
column, that allows the fractional distillation and concentration of
volatile compounds based on their volatility. The volumes of heads,
Table 1
Mean composition (g/100 g of pulp) of the pulps obtained from the red raspberry and
arbutus berry fruits.
Red raspberry Arbutus berry
Reducing sugars 4.17 ± 0.28 15.66 ± 1.20
Total protein (N × 6.25) 0.87 ± 0.12 3.18 ± 0.22
Moisture content 89.90 ± 1.24 71.30 ± 1.33
Ashes 0.41 ± 0.09 0.56 ± 0.15
pH 3.36 ± 0.04 3.50 ± 0.21
 E.A. González et al. / Food Research International 44 (2011) 1419–1426
hearts, and tails fractions of the two distillates depended on the
source of substrate from which they had been made. The first volume
of distillate (0.4 L in case of red raspberry and 0.2 L in case of arbutus
berry), corresponding to the beginning of the distillation procedure
when the temperature reached 70–85 °C, was removed as “head”. The
intermediate fraction called the “heart” (3.5 L in case of red raspberry
and 1.2 L in case of arbutus berry), the most important part of the
distilled fractions and that employed for spirit elaboration, was
obtained in the temperature range of between 85 and 95 °C and used
for volatile compounds determination. The last volume of distillate
(1.0 L in case of red raspberry and 1.3 L in case of arbutus berry)
obtained in the temperature interval between 95 and 99 °C was
removed as the “tail”.
2.7. Aromatic compounds determination
Volatile compounds present in the distilled “heart” fractions of red
raspberry (RBD) and arbutus berry (ABD) were determined by gas
chromatography on an Agilent 6890 (Agilent Technologies, Wald-
bronn, Germany) equipped with split/splitless injector, electronic
flow control (EFC) and a flame ionization detector (FID) as described
by López, Bollaín, Berstsch, and Orriols (2010).
According to this method, the compounds were separated on a
50 m × 0.32 mm I.D. × 0.2 μm df capillary column CP-WAX-57 CB
(Varian) linked to a capillary column deactivate (1 m × 0.32 mm I.
D.). The temperatures of the injector and detector were fixed at 220 °C
and 240 °C, respectively. The temperature program of the oven was as
follows: 40 °C for 3 min, a first linear ramp from 40 º C to 75 °C at 6 °C/
min and a second linear ramp from 75 °C to 210 °C at 9 °C/min.
Hydrogen was used as the carrier gas at a pressure of 130 kPa. The
injection mode was split with split ratio 1:20.
All of the volatile compounds were identified by comparing gas
chromatography retention times with those of 33 pure standard
compounds (including alcohols, aldehydes, acetates and esters) used
as references. For volatile compounds quantification, a 5 mL sample of
standard solution or distillate (RBD and ABD) was mixed with 50 μL of
internal standards solution (4-methyl-2-pentanol and 4-decanol) and
after agitation during 15 s, an aliquot (1 μL) of each mixture was
injected directly into the gas chromatograph. Calibration curves
(relative peak area versus concentration ratio of volatile compound/
internal standard) and all quantifications were performed by the
internal Standard method using Chemstation Rev.A.10.02 [1757]
Agilent Technologies.
All analyses were done in triplicate.
2.8. Reagents used as reference compounds
Pure deionised water was obtained from an Elix 3 purification
system (Millipore, Bedford, USA). Ethanol absolute (quality ACS-ISO)
was supplied by Merck (Darmstadt, Germany). Chemical standards
(alcohols, aldehydes, acetates and esters) were purchased from Merck
(Darmstadt, Germany), Fluka and Sigma Aldrich (Alcobendas, Madrid,
Spain) with the highest purity available.
A stock solution of volatile reference standards was prepared in
distilled water containing 45% (v/v) of ethanol, except acetaldehyde
solution which was prepared in water. The internal standard solutions
were prepared by dissolving 50 g of 4-methyl-2-pentanol and 1.3 g of
4-decanol in a 50% (v/v) mixture of distilled water and ethanol.
2.9. Statistical analysis
The data concerning mean concentration of fermentation products
and volatile compounds in the distillates obtained from fermented red
raspberry and arbutus berry were statistically analyzed by using the
software package SPSS Statistics 17.0 for Windows (Release 17.0.1;
SPSS Inc., Chicago, IL, 2008). A paired-samples t-test was conducted to
1421
determine whether significant differences (P b 0.05) existed between
the means obtained for the concentrations of fermentation products
and volatile compounds in the two distillates.
A cluster analysis was used to determine the similarity or
dissimilarity between our two distillates (RBD and ABD) and other
alcoholic beverages (Alonso et al., 2010; Diéguez et al., 2005; Dragone,
Mussatto, Oliveira, & Teixeira 2009; Silva, Malcata, & De Revel 1996,
Soufleros et al., 2004) by using their mean volatile composition as the
classification variables. The data were standardized before clustering,
to make all characteristics equally contribute to the discrimination
process. According to this procedure, both the magnitude and
variability of all mean concentrations of each volatile compound
were homogenized by transforming the original value of each variable
into a z-score, by using the following formula:
yi − y
zi =
sdy
where zi is the z score, yi is the original value of each variable, –
y is the
mean of all values of y, and sd is the standard deviation of that mean.
With this transformation, each variable has a mean of 0 and a standard
deviation of 1. The Euclidean distance was used as the distance
measure or similarity index and the average linkage (in the variant of
unweighted pair-group average) was used as the amalgamation
(linkage) method. Both the statistical analyses and the dendogram
plot were performed using the Cluster Analysis module of the
Statistica for Windows program, Release 5.1.
3. Results and discussion
3.1. Solid-state fermentations of the pulps by the wild microbiota
Some alcoholic beverages from different fruits of the forest are
commonly produced by submerged liquid fermentation of the fruit
juices (Darias et al., 2003). Although this fermentation procedure
requires a less complicated control than the solid-state fermentation of
the fruits, generally the latter method allows obtaining fermented
products with a high aromatic profile, which are composed by the
aromas from the raw material and those produced during the
fermentation process (Alonso et al., 2010). For this reason, the solid-
state fermentation of red raspberry and arbutus berry was spontane-
ously carried out with their native microbiota.
The results obtained in both spontaneous fermentations are shown
in Figs. 1 and 2, and in Table 2. As it can be observed, the final
concentrations of ethanol (0.15 g/100 g of pulp) and glycerol (0.13 g/
100 g of pulp) obtained in the red raspberry culture were only slightly
higher than those (0.13 and 0.08 g/100 g of pulp) obtained in the
arbutus berry culture (Fig. 2 and Table 2). In both cultures, a low
0.16 6
0.12
Ethanol
4
pH
0.08
2
0.04
0 0
Reducing Sugars
0.15
Glycerol
4
0.10
2
0.05
0 0
0 20 40 60 0 20 40 60 80
Time (h)
Fig. 1. Time course of the spontaneous solid-state fermentation of red raspberry pulp.
Ethanol, glycerol and reducing sugars are expressed in g/100 g of fermentation medium.
1422 E.A. González et al. / Food Research International 44 (2011) 1419–1426

0.20 4 3 4

0.15 3 3

Ethanol
2
Ethanol

pH
pH
0.10 2 2
1
0.05 1 1

0 0 0 0

Reducing Sugars
Reducing Sugars
0.075 15 0.075

Glycerol
4
Glycerol

0.050 10 0.050
2
0.025 5 0.025

0 0 0 0
0 20 40 60 0 20 40 60 80 0 20 40 60 0 20 40 60 80
Time (h) Time (h)

Fig. 2. Time course of the spontaneous solid-state fermentation of arbutus berry pulp. Fig. 3. Time course of the solid-state fermentation of the thermally treated red
Ethanol, glycerol and reducing sugars are expressed in g/100 g of fermentation medium. raspberry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars
are expressed in g/100 g of fermentation medium.

reducing sugars consumption was observed. Thus, only the 14.8% (in
case of red raspberry) and the 3.2% (in case of arbutus berry) of the
initial reducing sugars concentration were consumed (Table 2).
The ethanol yields from reducing sugars consumed (YEt/RSc in g/g)
were calculated as 0.255 (in case of red raspberry) and 0.246 (in case
of arbutus berry), which correspond to 50 and 48% of the theoretical
ethanol yield from glucose (0.511 g/g). In the same way, the ethanol
productivities (PEt) obtained at the end of the incubations in the red
raspberry culture (0.0023 g/100 g of pulp/h) and in the arbutus berry
culture (0.0020 g/100 g of pulp/h) were very similar (Table 2).
Surprisingly, in the arbutus berry culture (Fig. 2), ethanol
production was detected only after 61 h of fermentation, probably
due to the high viscosity observed in the fruit pulp, justly before
starting the spontaneous solid-state fermentation. This high viscosity
could be related with the high concentration in pectins, between 3.0
and 4.6%, as dry weight (Alarcão-E-Silva et al., 2001), as well as with
the low water content (71.3%) in the arbutus berry pulp (Table 1).
Thus, the high viscosity present in the arbutus berry pulps could
interfere with the mass transfer, thus limiting the growth of the
indigenous yeasts and, consequently, ethanol production (Alonso
et al., 2010).
A similar fermentation procedure was carried out by other
researchers (Cavaco et al., 2007) to reproduce the little known
fermentation phase of the production of Aguardente de Medronho, a
distillate very appreciated in Portugal. In this case, the arbutus berry
pulp was fermented by its wild microbiota during 36 days (as it is
performed in the farms), to gain a better understanding of the
fermentation process. This study showed that yeasts were the main
organism responsible for the fermentation of the fruits, because the
counts of both lactic acid and acetic acid bacteria were lower than
10 CFU/mL. In addition, the yeast population stops to grow after
8 days of fermentation coinciding with a decrease in the sugars
(sucrose, glucose and fructose) concentration. However, ethanol
production stopped after 16 days of fermentation. These observations
indicate that the duration of the fermentation process could be
reduced from 28 or 35 days (which are the fermentation times used in
the farm) to 16 days, in order to reduce the production cost and
increase the ethanol productivity of this distillate.
Therefore, in an attempt for increasing the ethanol productivities
in the fermentations of the red raspberry and arbutus berry, the
following study was focused on the use of a high ethanol-producing
yeast to ferment the pulps. This approach will allow the production of
high levels of ethanol in short incubation times.
3.2. Solid-state fermentations of the thermally treated pulps by
S. cerevisiae IFI83
The results obtained in the inoculated solid-state fermentations of
the thermally treated pulps from red raspberry and arbutus berry are
presented in Figs. 3 and 4, respectively. From the detailed observation
of both cultures, it can be noted that the amounts of sugars consumed
by S. cerevisiae IFI83 in both cultures were considerably higher
(P b 0.05) than those consumed by the wild microbiota of both fruits in
the spontaneous cultures (Table 2). In this way, the sugar consump-
tion percentages corresponded to 94.1% (in case of red raspberry) and
58.2% (in case of arbutus berry) of initial levels (Figs. 3 and 4).
The final concentrations of ethanol obtained (1.81 g/100 g of pulp,
in case of red raspberry) and 3.03 g/100 g of pulp (in case of arbutus
berry) were respectively, 12 and 23 times higher (P b 0.05) than those
obtained in the spontaneous fermentations (Table 2). In addition, the
4 4
3 3
Ethanol

pH

Table 2 2 2
Fermentation parameters obtained in the solid-state fermentations of red raspberry
(RB) and arbutus berry (AB) pulps. 1 1

Spontaneous Inoculated fermentations 0 0

Reducing Sugars

fermentation
Thermally Nonthermally 0.45 15
Glycerol

treated pulp treated pulp

0.30 10
Parameters RB AB RB AB RB AB
0.15 5
RSc (g/100 g of pulp) 0.62 0.48 3.95 8.87 3.97 9.04
Et (g/100 g of pulp) 0.15 0.13 1.81 3.03 1.78 3.20 0 0
Glyc (g/100 g of pulp) 0.13 0.08 0.05 0.48 0.06 0.48 0 20 40 60 0 20 40 60 80
YEt/RSc 0.255 0.246 0.459 0.342 0.446 0.354
Time (h)
PEt (g/100 g of pulp/h) 0.0023 0.0020 0.029 0.050 0.028 0.052

RSc are the reducing sugars consumed. Glyc is the glycerol concentration. YEt/RSc is the Fig. 4. Time course of the solid-state fermentation of the thermally treated arbutus
ethanol (Et) yield from reducing sugars (as glucose) consumed. PEt is the ethanol berry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars are
productivity (ethanol produced per hour of fermentation). expressed in g/100 g of fermentation medium.
 E.A. González et al. / Food Research International 44 (2011) 1419–1426 1423

4 4
YEt/RSc values obtained (0.459 and 0.342 in the fermentations on red
raspberry and arbutus berry), were respectively, 90 and 67% of the 3 3

Ethanol
above mentioned theoretical ethanol yield from glucose. Thus, the

recourse of inoculating the fruits with a relatively high concentration
of the ethanol-producing yeast (S. cerevisiae IFI83) led to a more
efficient utilization of the reducing sugars for ethanol production than
pH
2 2
1 1
0 0

Reducing Sugars
did the indigenous microbiota of the fruits in the spontaneous
0.45 15

fermentations (Table 2).
On the other hand, the higher ethanol productivities obtained
(0.029 and 0.050 g/100 g of pulp/h, in the red raspberry and arbutus
berry cultures) indicated that the inoculated fermentations were
more productive than the spontaneous fermentations.
Although the thermally treated pulps can be efficiently fermented
by S. cerevisiae IFI83 for ethanol production, the fermented pulps
presented non-disagreeable cooked and less fruity odors, as it was
observed before with black mulberry and black currant (Alonso et al.,
2010). For this reason, the following solid-state cultures with S.
cerevisiae IFI83 were carried out by using the nonthermally treated
pulps as fermentation substrates.
3.3. Solid-state fermentations of the nonthermally treated pulps by
S. cerevisiae IFI83
The kinetic profiles of the solid-state fermentation of the two
nonthermally treated pulps by S. cerevisiae IFI83 are shown in Figs. 5
and 6. In both cultures, the amounts of reducing sugars consumed, the
levels of ethanol and glycerol produced as well as the YEt/RSc and PEt
values were similar to those levels obtained in the inoculated solid-
state fermentation of the thermally treated pulps (Table 2).
These results indicated that the thermal treatment of the fruits
neither affects the final concentrations nor the yield and productivity
of ethanol obtained. However, with the use of the nonthermally
treated pulps, the fermented products did not present undesirable
or unpleasant odors as occurred in the previous two cultures (Figs. 3
and 4).
Therefore, the distillation and the aroma compounds identification
in the distillates were carried out from nonthermally treated pulps
fermented by solid-state cultures with S. cerevisiae IFI83.
3.4. Volatile compounds present in the red raspberry and arbutus berry
distillates
Glycerol
0.30 10
0.15 5
0 0
0 20 40 60 0 20 40 60 80
Time (h)
Fig. 6. Kinetics of the solid-state fermentation of the nonthermally treated arbutus
berry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars are
expressed in g/100 g of fermentation medium.
Council for fruit distillates (Reg. EEC 110/2008), because there are not
legal restrictions concerning fruit distilled beverages in Spain.
In this way, the mean alcoholic concentration detected in the RBD
(41.3%) and ABD (44.3%) were within the limits of acceptability [from
37.5 to 86.0% (v/v)] given by the European Council (Reg. EEC 110/
2008).
Methanol is a highly toxic product whose inhalation or ingestion
can cause blindness and eventually death (Geroyiannaki et al., 2007).
This compound is generated by the enzymatic action of the pectin-
methylesterases that catalyze the hydrolysis of the esterified
methoxyl groups of the pectin polymer present in crushed fruits
(Geroyiannaki et al., 2007; Silva et al., 1996; Soufleros et al., 2004).
Thus, the presence of methanol in distilled spirits is directly linked to
the pectin content of the raw material (Bindler, Voges, & Laugel 1988).
From our results (Table 3), it can be noted that RBD had a mean
methanol concentration (113.9 g/hL aa) almost three times lower
(P b 0.05) than that of the ABD (320.5 g/hL aa). However, both samples
contain methanol levels lower than the legal maxima levels of 1200 g/
hL aa (for a red raspberry spirit) or 1000 g/hL aa (for an arbutus berry
spirit), adopted by the Regulating Commission (Reg. EEC 110/2008).
This indicates that both fruits were adequately manipulated and that
their distillates were obtained by using an adequate distillation
procedure (Silva et al., 1996; Soufleros et al., 2004).

The concentrations of the main volatile compounds in the “heart” Table 3

fractions of the red raspberry (RBD) and arbutus berry (ABD) Mean concentration (g/hL aa) of volatile compounds present in the “heart” fractions of
red raspberry (RBD) and arbutus berry (ABD) distillates.
distillates are shown in Table 3. Our results were compared with
the maximum and minimum concentrations fixed by the European no. Compound RBD ABD

1 Ethanol (% v/v) 41.3 ± 1.38a 44.3 ± 1.30a

3 4 2 Methanol 113.9 ± 1.44a 320.5 ± 3.48b
3 1-propanol 36.2 ± 0.15a 41.0 ± 0.88b
3
Ethanol

2 4 1-butanol 0.5 ± 0.07a 0.8 ± 0.09b

pH

2 5 2-butanol 0.2 ± 0.008a nd

1 6 2-methyl-1-propanol (isobutyl alcohol) 33.4 ± 0.74a 36.0 ± 1.85a
1 7 2-methyl-1-butanol 11.8 ± 1.16a 13.8 ± 0.73a
8 3-methyl-1-butanol 67.9 ± 1.41a 78.7 ± 1.77b
0 0
Reducing Sugars

Total alcohols (3–8) 150.0 ± 3.54a 170.3 ± 5.32b

0.075 9 Allyl alcohol 0.02 ± 0.001a nd
Glycerol

4 10 1-hexanol 1.1 ± 0.04a 1.2 ± 0.21a

0.050 11 Benzyl alcohol 2.5 ± 0.13a 1.4 ± 0.07b
2 12 2-phenyl ethanol nd nd
0.025
13 Ethyl acetate 37.8 ± 0.34a 40.7 ± 3.16b
0 0 14 Ethyl lactate (ethyl 2-hydroxypropanoate) 0.3 ± 0.06a 0.3 ± 0.09a
0 20 40 60 0 20 40 60 80 15 Acetaldehyde 4.4 ± 0.17a 32.7 ± 1.88b
16 Acetal 4.0 ± 0.25a 20.5 ± 1.33b
Time (h)
Total volatile substances (3–15) 200.1 ± 4.53a 267.1 ± 12.06b

Fig. 5. Kinetics of the solid-state fermentation of the nonthermally treated red Means within the same row, followed by the same letter are not significantly different
raspberry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars at 95% confidence. BMD and BCD are the black mulberry and the black currant
are expressed in g/100 g of fermentation medium. distillates. nd, not detected.
1424 E.A. González et al. / Food Research International 44 (2011) 1419–1426
Higher alcohols, the group of highest concentration in distilled
alcoholic beverages, are generally regarded as important flavor
compounds with a great influence in the quality of a distillate
(Diéguez et al., 2005; Dragone et al., 2009; Lachenmeier, Haupt, &
Schulz 2008; Silva & Malcata 1999; Soufleros et al., 2004). Although
the European Council (Reg. EEC 110/2008) does not, at the present
time, fix maximum and minimum values for the higher alcohols
content in fruit distillates, it is considered that a total concentra-
tion ≥ 350 g/hL aa in distilled beverages is indicative of a poor quality
(Rodríguez & Mangas 1996). Since the concentrations of higher
alcohols in both the RBD (150.0 g/hL aa) and ABD (170.3 g/hL aa)
samples (Table 3) were lower than 350 g/hL aa, it can be concluded
that these two distillates have a good quality.
The concentrations of 1-propanol were higher (P b 0.05) than those
of 1-butanol in RBD and ABD (Table 3). Both alcohols are considered to
be strongly odor compounds and their presence at high concentrations
in distillates are indicative of bacterial spoilage during storage of the
material before distillation (Apostolopoulou, Flouros, Demertzis, &
Akrida-Demertzi 2005; Diéguez et al., 2005). However, the concentra-
tions of 1-propanol and 1-butanol in RBD (149.5 and 2.1 mg/L,
respectively) and ABD (181.6 and 3.5 mg/L, respectively) did not
surpass the perception thresholds of 800 and 450 mg/L for both
compounds (De Rosa & Castagner 1994). These results suggest that
the fermentations of red raspberry and arbutus berry pulps and their
storage were carried out in adequate conditions (Dragone et al., 2009).
The overall quality of distillates is clearly reduced when 2-butanol
is present in them even at low concentrations (Silva & Malcata 1998;
1999), mainly because this product accounts for unpleasant aromas
and flavors (Bertrand & Sukuta 1976). This alcohol could be originated
from butane-2,3-diol by the action of lactic acid bacteria (Manitto,
Chialva, & Rinaldo 1994). Thus, high concentrations of 2-butanol in
distilled alcoholic beverages could be indicative of bacterial spoilage
in the raw materials used for distillation (Speranza, Corti, Fontana, &
Manitto 1997). Since the concentrations of 2-butanol in the ABD (not
detected) and RBD (0.2 g/hL aa) samples was lower than the value
(30 g/hL aa) considered as deleterious to the quality of the product
(Diéguez et al., 2005), it can be considered that both distillates are of
good quality according to this criterion.
2-Methyl-1-propanol is produced as the transamination product
of the amino acid precursor valine (Zoecklein, Fugelsang, Gump, &
Nury 1995). This compound as well as isobutanol, 2-methyl-l-butanol,
3-methyl-butanol and hexanol have been claimed to contribute
favorable keynotes to spirits (Silva, Macedo, & Malcata 2000). In our
two samples, the concentrations of 2-methyl-1-propanol (Table 3) were
33.4 g/hL aa, equivalent to 137.9 mg/L (in case of RBD) and 36.0 g/hL aa,
equivalent to 159.5 mg/L (in case of ABD). Although 2-methyl-1-
propanol is present in both samples at concentrations lower than the
perception threshold of 200 mg/L (De Rosa & Castagner 1994), this
would not affect the quality of both distillates because the Galician orujo
spirit from Godello (Diéguez et al., 2005), an alcoholic beverage with a
recognized high taste and bouquet, also had a 2-methyl-1-propanol
concentration (195 mg/L) lower than 200 mg/L.
Amyl alcohols (2-methyl-1-butanol and 3-methyl-1-butanol),
which have an aromatic description of alcoholic, sweet and choking
are formed during fermentation by deamination and decarboxylation
reactions from leucine and iso-leucine, respectively (Boulton, Singleton,
Bisson, & Kunkee 1996; Kana, Kanellaki, Kouinis, & Koutinas 1988). The
concentrations of 2-methyl-1-butanol and 3-methyl-1-butanol were
slightly higher (P N 0.05) and higher (P b 0.05) in the ABD (61.1 and
348.6 mg/L) than in RBD (48.7 and 280.4 mg/L). Thus, only the
concentrations of 2-methyl-1-butanol in both samples were lower
than the perception threshold of 65 mg/L reported for both compounds
(De Rosa & Castagner 1994). Since low concentrations of amyl alcohols
(2-methyl-1-butanol and 3-methyl-1-butanol) are associated with
light-bodied orujo spirits (Diéguez et al., 2005; Silva et al., 1996), it
can be suggested that the ABD has a better body than the RBD.
Presence of allyl alcohol in distillates is related to a deficient storage
of the raw material (De Rosa & Castagner 1994). Since this product were
not detected in ABD and was detected at a low concentration in RBD
(0.02 g/hL aa), it could be considered that the arbutus berry and red
raspberry fruits were manipulated under adequate storage conditions.
Presence of 1-hexanol, a typical heart product (Flouros,
Apostolopoulou, Demertzis, & Akrida-Demertzi 2003), has a positive
influence on the aroma of a distillate when occurs in concentrations
up to 20 mg/L (Apostolopoulou et al., 2005; Flouros et al., 2003). At
high levels, this product, having an aromatic description of
“coconut-like”, “harsh” and “pungent”, can produce some defects
(“green flavor”) in the distillates (Falqué, Fernández, & Dubourdieu
2001). Taking into account the low concentrations of 1-hexanol
found in RBD (1.1 g/hL aa, equivalent to 4.5 mg/L) and ABD (1.2 g/
hL aa, equivalent to 5.3 mg/L), it could be considered that the
presence of this compound in our samples did not affect negatively
their flavors.
2-Phenylethanol is characterized as a tail component, so its
concentration in the heart fraction of distillates would be very low
(Apostolopoulou et al., 2005; Flouros et al., 2003). This product, which
is an aroma carrier that may contribute to the floral nuance of
alcoholic beverages (Duarte, Dias, et al., 2010), was not detected in
both the RBD and ABD samples (Table 3). This suggests that the heart
cut in the fractional distillation of the fermented fruits was adequately
performed.
Benzyl alcohol is an aromatic compound that contributes to the
flowery and sweet-like odors of alcoholic beverages (Falqué et al.,
2001; Perestrelo, Fernandes, Albuquerque, Marques, & Câmara 2006).
Therefore, presence of this compound in the RBD (2.5 g/hL aa) and
ABD (1.4 g/hL aa) could be considered as a positive characteristic for
both distillates.
High amounts of ethyl acetate in distillates could be indicative of
acetic bacterial spoilage (Dragone et al., 2009; Silva & Malcata 1998;
1999) or the result of an incorrect separation of the head fraction
during distillation (Diéguez et al., 2005). Presence of this compound
has a significant contribution on the organoleptic characteristics of
distilled alcoholic beverages. At concentrations lower than 150 mg/L
in the distillates, this ester contributes to a pleasant aroma with fruity
properties. Contrarily, when its concentration exceeds 150 mg/L,
ethyl acetate gives a vinegary character and adds spoilage notes to the
alcoholic beverages (Apostolopoulou et al., 2005; Dragone et al., 2009;
Rodríguez, Picinelli, & Mangas 2010). The concentrations of this
compound in the RBD (37.8 g/hL aa, equivalent to 156.1 mg/L) and
ABD (40.7 g/hL aa, equivalent to 180.3 mg/L) samples (Table 3) were
higher than 150 mg/L. This suggests that the ethyl acetate concen-
tration in both distillates was not at a suitable level to confer a
pleasant flavor. However, presence of this compound at concentra-
tions much higher than 150 mg/L has been detected in four Galician
orujo spirits (Diéguez et al., 2005): Mencia (565 mg/L), Godello
(338 mg/L), Albariño (453 mg/L) and Treixadura (567 mg/L) or in
different bagaceiras (in the range of 50 and 530 mg/L) (Silva et al.,
1996), which are commercialized alcoholic beverages with a high
quality.
At low concentrations, ethyl lactate contributes to the stabilization
of the distillate flavor and softens the harsh flavor characteristics
(Apostolopoulou et al., 2005). However, at high concentrations, this
compound affects negatively the organoleptic quality of the distillates
(Manitto et al., 1994). Since this ester was detected at a low
concentration (0.3 g/hL aa) in both the RBD and ABD samples, it
could be considered that both the red raspberry and arbutus berry
pulps were fermented under adequate conditions and without
intervention of unwanted lactic acid bacteria (Alonso et al., 2010;
Falqué et al., 2001).
Acetaldehyde, a potent flavor compound commonly present in
many alcoholic beverages (Geroyiannaki et al., 2007), is produced by
decarboxylation of pyruvate during the alcoholic fermentation by
 E.A. González et al. / Food Research International 44 (2011) 1419–1426
yeast (Pronk, Steensma, & van Dijken 1996). However, this product
may also be formed by the metabolic activity of lactic acid bacteria or
acetic acid bacteria (Pieper, Rau, Eller, & Volz 1987). The concentra-
tion of this product increases commonly during aging of alcoholic
beverages as a consequence of the chemical oxidation of ethanol
(Geroyiannaki et al., 2007). At low levels, acetaldehyde gives a
pleasant fruity aroma, but at high concentrations it possesses a
pungent irritating odor (Miyake & Shibamoto 1993). Since this
compound is generally considered as a source of carcinogenicity in
alcoholic beverages, its presence in the distillates should be
diminished as far as possible to avoid a safety risk to consumers as
indicated by Lachenmeier and Sohniusa (2008). These researchers
proposed a model calculation to estimate approximately the residual
salivary acetaldehyde concentration in the beverage/saliva mixture
after drinking of a swallow of any alcoholic beverage. By using
this method and taking into account the mean acetaldehyde con-
centrations in RBD (4.4 g/hL aa, equivalent to 18.2 mg/L) and in ABD
(32.7 g/hL aa, equivalent to 144.9 mg/L), the residual acetaldehyde
concentrations in the saliva after drinking of a swallow of these
beverages could be on average 372 μM for RBD and 2963 μM for
ABD. Both values are above the acetaldehyde salivary concentrations
of 40–200 μM, previously considered as potentially carcinogenic
(Lachenmeier & Sohniusa, 2008; Lachenmeier 2008).
Formation of acetals reduces the content of free aldehydes in
distillates and consequently, the pungent and sour odors caused by
the latter compounds may be efficiently smoothed down (Silva et al.,
2000) and substituted by the pleasant and fruity odors of acetals
(Simon, Meersman, Piggott, & Conner 1997). In our samples (Table 3),
the amount of acetal detected in RBD (4.0 g/hL aa) was significantly
lower (P b 0.05) than that detected in ABD (20.5 g/hL aa), suggesting
that the latter sample has the most pleasant aroma.
The total volatile compounds content in the ABD (267.1 g/hL aa)
and RBD (200.1 g/hL aa) were respectively higher and slightly higher
than the minimum level of 200 g/hL aa, fixed by the European Council
Regulation 110/2008 (Regulation EEC110/2008) for fruit distillates.
Therefore, mainly for RBD, it is necessary to adjust the time for
removal of “head” and “tail” fractions, in order to obtain a distillate
with a higher concentration of volatile compounds (Alonso et al.,
2010).
3.5. Comparison of the volatile composition of the RBD and ABD with
those of other commercialized alcoholic beverages
Hierarchical cluster analysis was carried out to compare the mean
volatile composition of RBD and ABD with those of nine alcoholic
beverages obtained from different raw materials (fruits of the forest,
grape marc and whey) and with the use of different storage,
fermentation and distillation conditions. The nine distillates used in
the comparison were: i) BCD, BMD and BBD, which were respectively
obtained from fermented black currant, black mulberry (Alonso et al.,
2010) and blackberry (Soufleros et al., 2004), ii) the Portuguese
bagaceiras: Bag (Silva et al., 1996) and four commercial Galician orujo
spirits (Diéguez et al., 2005), including Albariño: Al, Mencia: Me,
Godello: Gd and Treixadura: Tr, obtained from grape marc, and iii) a
distillate obtained from fermented whey (WD) (Dragone et al., 2009).
In the hierarchy (Fig. 7), two main groups composed were
observed: one grouping the five distillates obtained from fruits of
the forest (ABD, RBD, BCD, BMD and BBD) and the other one grouping
the five distillates obtained from grape marc (Al, Go, Tr, Me and Bag).
In addition, each independent group was formed by four subclusters
with different Euclidean distances (ED). Thus, in the first group the
ABD and RBD samples merge to form the subcluster 1 with an ED of
1.48. Subsequently, BCD associated with subcluster 1 to form the
subcluster 2 (ED = 2.35) and BMD were associated with the latter
subcluster to form the subclusters 4 (ED = 3.49). Finally, subcluster 4
and BBD sample form the subcluster 8 (ED = 6.12). On the other hand,
1425
Fig. 7. Dendogram of cluster analysis for the eleven alcoholic beverages. Al: Albariño, Me:
Mencia, Gd: Godello, Tr: Treixadura, Bag: Portuguese bagaceiras, WD: whey distillate, BBD:
blackberry distillate, BMD: black mulberry distillate, BCD: black currant distillate, RBD: red
raspberry distillate and ABD: arbutus berry distillate. The standardized mean concentra-
tions of the volatile compounds of each distillate were used as classification variables.
the second group was formed by the subcluster 3 composed by the
samples of Albariño and Godello (ED = 2.56), the subcluster 5
(Treixadura and subcluster 3, ED = 3.74), subcluster 6 (Mencia and
subcluster 5, ED = 4.36) and the subcluster 7 (bagaceiras and
subcluster 6, ED = 6.00). The WD sample was the most different
distilled alcoholic beverage because it merges with the group formed
by the other ten distillates with the highest ED value (ED = 7.30).
These results suggested that the type of raw material was the main
factor influencing the group formation. With regard to this, the
Albariño, Godello and Treixadura spirits obtained from white grapes
are successively grouped to form the clusters 3 and 5, and finally the
Mencia spirit obtained from red grapes, was associated with the
cluster 5 to form the cluster 6.
However, formation of the independent first and second groups
seems to be influenced by other factors. For example, the samples of
RBD, BCD, ABD and BMD (subcluster 4) were obtained by using the
same fermentation and distillation procedures in our laboratory, but
the mean compositions of the fruits used as raw material were found
to be different (Table 1; Alonso et al., 2010). On the other hand, the
BBD sample (Soufleros et al., 2004), which was associated with
subcluster 4, was obtained by using procedures of fermentation
(spontaneous fermentation) and distillation different than those used
to obtain our four distillates (RBD, BCD, ABD and BMD). In addition,
although both the BBD (Soufleros et al., 2004) and BMD (Alonso et al.,
2010) were obtained from the same raw material (Morus nigra L.), the
different origin of the fruits (Portugal and Spain, respectively)
probably had a great influence on the different mean volatile
composition obtained in both distillates. In the same way, the four
orujo spirits (Al, Go, Tr and Me), which form the subcluster 6, are
produced from red and white grape varieties by using the same basic
process, but the system and time of storage as well as the
fermentation and distillation procedures were different (Diéguez et
al., 2005).
4. Conclusions and future prospects
In both the red raspberry and arbutus berry distillates, the
concentrations of methanol, ethanol and volatile substances were in
accordance with the specifications fixed by the European Council
(Regulation 110/2008) for fruit distillates.
The results of the present study contribute to the understanding,
improvement and reproduction of the solid-state fermentation
processes of red raspberry and arbutus berry to produce two high-
quality distillates (RBD and ABD). The production of these two
alcoholic beverages could allow an effective preservation and
valorization of both fruits, and their posterior commercialization
could greatly increase the farmer income in Galicia.
1426 E.A. González et al. / Food Research International 44 (2011) 1419–1426
In this way, further studies based on the solid-state fermentation
of red raspberry and arbutus berry using polyurethane foams as
support material are being assayed in our laboratory in order to select
the most adequate fermentation procedure for producing the two
distillates and make them more competitive.
Acknowledgment
The research presented in this paper was financially supported by
the Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA), Spain (project RTA2005-00165-C02-00).
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