Solid-state fermentation of red raspberry (Rubus ideaus L.) and arbutus berry (Arbutus unedo, L.) and characterization of their distillates

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 b

Departamento de Química Analítica y Alimentaria. Facultad de Ciencias, Universidad de Vigo, Campus de Ourense, As Lagoas s/n, 32004, Ourense, Spain 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

Article history: Received 29 November 2010 Accepted 16 February 2011 Keywords: Alcoholic beverages arbutus berry Red raspberry Ethanol Solid-state fermentation Volatile compounds

a b s t r a c t The aim of the present study was to obtain two distilled alcoholic beverages from red raspberry and arbutus berry by solid-state fermentation and subsequent distillation of the fermented fruits. The mean 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 200 g/hL aa) fixed by the European Council (Regulation 110/2008) for fruit distillates. In addition, the mean concentrations of methanol in the two alcoholic beverages (113.9 g/hL aa in case of red raspberry, and 320.5 g/hL aa in case of arbutus berry) were much lower than the maximum levels of acceptability that the aforementioned regulation fixed for red raspberry (1200 g/hL aa) and arbutus berry (1000 g/hL aa) distillates. 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: [email protected] (N.P. Guerra). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.02.032

value products (e.g. distillates from fruits of the forest) and consequently, 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 (AlarcãoE-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

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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 infrastructures 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 (3.5Dinitrosalicylic 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 nonthermally 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.

Reducing sugars Total protein (N × 6.25) Moisture content Ashes pH

Red raspberry

Arbutus berry

4.17 ± 0.28 0.87 ± 0.12 89.90 ± 1.24 0.41 ± 0.09 3.36 ± 0.04

15.66 ± 1.20 3.18 ± 0.22 71.30 ± 1.33 0.56 ± 0.15 3.50 ± 0.21

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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:

2.7. Aromatic compounds determination

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

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 solidstate fermentation of red raspberry and arbutus berry was spontaneously 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 6

0.16 0.12

4

pH

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.

yi − y sdy

Ethanol

2.8. Reagents used as reference compounds

zi =

0.08 2

0.04 0

Glycerol

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, Waldbronn, 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.

0

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0

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60

0 80

Reducing Sugars

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”.

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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.

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0.05

1

0

0

0.075

15

0.050

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0 40

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0

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40

0 80

60

1

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0

0.075

4

0.050 2

0.025 0 0

20

40

60

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

Inoculated fermentations Thermally treated pulp

Nonthermally treated pulp

RB

RB

RB

0.62 0.15 0.13 0.255 0.0023

0.48 0.13 0.08 0.246 0.0020

60

0 80

3.95 1.81 0.05 0.459 0.029

AB 8.87 3.03 0.48 0.342 0.050

3.97 1.78 0.06 0.446 0.028

AB 9.04 3.20 0.48 0.354 0.052

RSc are the reducing sugars consumed. Glyc is the glycerol concentration. YEt/RSc is the ethanol (Et) yield from reducing sugars (as glucose) consumed. PEt is the ethanol productivity (ethanol produced per hour of fermentation).

(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 consumption 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

Ethanol

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

RSc (g/100 g of pulp) Et (g/100 g of pulp) Glyc (g/100 g of pulp) YEt/RSc PEt (g/100 g of pulp/h)

40

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

Glycerol

Fig. 2. Time course of the spontaneous solid-state fermentation of arbutus berry pulp. Ethanol, glycerol and reducing sugars are expressed in g/100 g of fermentation medium.

AB

20

Time (h)

Time (h)

Parameters

0

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Reducing Sugars

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Glycerol

4

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0.20

Reducing Sugars

Glycerol

Ethanol

1422

Time (h) Fig. 4. Time course of the solid-state fermentation of the thermally treated arbutus berry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars are expressed in g/100 g of fermentation medium.

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 solidstate 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 The concentrations of the main volatile compounds in the “heart” fractions of the red raspberry (RBD) and arbutus berry (ABD) distillates are shown in Table 3. Our results were compared with the maximum and minimum concentrations fixed by the European

3 2

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pH

0.075

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1 0

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0.050 2

0.025 0 0

20

40

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0

20

40

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0 80

Reducing Sugars

Ethanol

2

Glycerol

3

Time (h) Fig. 5. Kinetics of the solid-state fermentation of the nonthermally treated red raspberry pulp inoculated with S. cerevisiae IFI83. Ethanol, glycerol and reducing sugars are expressed in g/100 g of fermentation medium.

4

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1423

Reducing Sugars

Glycerol

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 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 did the indigenous microbiota of the fruits in the spontaneous 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.

Ethanol

E.A. González et al. / Food Research International 44 (2011) 1419–1426

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 pectinmethylesterases 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).

Table 3 Mean concentration (g/hL aa) of volatile compounds present in the “heart” fractions of red raspberry (RBD) and arbutus berry (ABD) distillates. no.

Compound

RBD

ABD

1 2 3 4 5 6 7 8

Ethanol (% v/v) Methanol 1-propanol 1-butanol 2-butanol 2-methyl-1-propanol (isobutyl alcohol) 2-methyl-1-butanol 3-methyl-1-butanol Total alcohols (3–8) Allyl alcohol 1-hexanol Benzyl alcohol 2-phenyl ethanol Ethyl acetate Ethyl lactate (ethyl 2-hydroxypropanoate) Acetaldehyde Acetal Total volatile substances (3–15)

41.3 ± 1.38a 113.9 ± 1.44a 36.2 ± 0.15a 0.5 ± 0.07a 0.2 ± 0.008a 33.4 ± 0.74a 11.8 ± 1.16a 67.9 ± 1.41a 150.0 ± 3.54a 0.02 ± 0.001a 1.1 ± 0.04a 2.5 ± 0.13a nd 37.8 ± 0.34a 0.3 ± 0.06a 4.4 ± 0.17a 4.0 ± 0.25a 200.1 ± 4.53a

44.3 ± 1.30a 320.5 ± 3.48b 41.0 ± 0.88b 0.8 ± 0.09b nd 36.0 ± 1.85a 13.8 ± 0.73a 78.7 ± 1.77b 170.3 ± 5.32b nd 1.2 ± 0.21a 1.4 ± 0.07b nd 40.7 ± 3.16b 0.3 ± 0.09a 32.7 ± 1.88b 20.5 ± 1.33b 267.1 ± 12.06b

9 10 11 12 13 14 15 16

Means within the same row, followed by the same letter are not significantly different at 95% confidence. BMD and BCD are the black mulberry and the black currant distillates. nd, not detected.

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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 concentration ≥ 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 concentrations 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-1propanol 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 concentration in both distillates was not at a suitable level to confer a pleasant flavor. However, presence of this compound at concentrations 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

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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 concentration 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 concentrations 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,

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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 concentrations 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 highquality 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.

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