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Yeast species associated with the spontaneous fermentation of cider
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 24 (2007) 25–31 www.elsevier.com/locate/fm
Yeast species associated with the spontaneous fermentation of cider Bele´n Sua´rez Vallesa, Rosa Pando Bedrin˜anaa,, Norman Ferna´ndez Tasco´na, Amparo Querol Simo´nb, Roberto Rodrı´ guez Madreraa a
A´rea de Tecnologı´a de los Alimentos, Servicio Regional de Investigacio´n y Desarrollo Agroalimentario (SERIDA) 33300, Villaviciosa, Asturias, Spain b Departamento de Biotecnologı´a, Instituto de Agroquı´mica y Tecnologı´a de los Alimentos (CSIC) 46100, Valencia, Spain Received 1 February 2006; received in revised form 27 March 2006; accepted 1 April 2006 Available online 21 April 2006
Abstract This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by nonSaccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cider yeasts; Spontaneous fermentation; PCR-RFLP
1. Introduction The fermentation of apple must is a complex microbial reaction involving the sequential development of various species of yeasts and bacteria (Beech, 1972; Salih et al., 1988; Cabranes et al., 1990; Duen˜as et al., 1994). Among these micro-organisms, yeasts are primarily responsible for alcoholic fermentation. Thus, the different yeast species developed during fermentation and their dynamics and frequency of appearance determine the taste and flavour characteristics of products (Beech and Davenport, 1970; Mafart, 1986; Le Quere´ and Drilleau, 1993; Cabranes et al., 1997; Sua´rez et al., 2005). In Asturias (Spain), natural cider is produced by the spontaneous fermentation of apple juice by yeasts originated from fruit and cider making equipment. This type of fermentation is of particular interest to ascertain the yeast Corresponding author. Tel.: 34 985 89 00 66; fax: 34 985 891 854.
E-mail address: [email protected] (R.P. Bedrin˜ana). 0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2006.04.001
species associated with the fermentation processes. Preliminary studies about the population dynamic have shown that the genus Saccharomyces is usually predominant during alcoholic fermentation, while the non-Saccharomyces genera, such as Kloeckera, Candida, Pichia, Hansenula, Hanseniaspora and Metschnikowia mainly grow during the first stages of the process (Michel et al., 1988; Cabranes, 1994). On the other hand, several factors such as geographic location, climatic conditions, apple varieties and the cider making technology can influence the diversity of yeasts present in must (Poulard et al., 1985; Cabranes et al., 1990; Mangas et al., 1994; del Campo et al., 2003). Traditionally, the identification and characterization of yeast species have been based on morphological traits and their physiological capabilities (Barnett et al., 1990). The conventional methodology for yeast identification requires evaluation of tests in a complex, laborious and timeconsuming process. In the last decade, microbial identification has undergone a revolutionary change by the introduction of PCR-based methodologies (Ness et al.,
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1993; Paffetti et al., 1995; de Barros Lopes et al., 1996). In this sense, one of the most successful methods for yeast species identification is the restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS) (White et al., 1990; Guillamo´n et al., 1998; Esteve-Zarzoso et al., 1999, 2001; Ferna´ndez-Espinar et al., 2000; Sabate et al., 2002). The aim of this study was to identify the nonSaccharomyces and Saccharomyces biota present in apple musts at different phases of fermentation in one cellar during two consecutive years. Also, the influence of the cider-making systems on the diversity and dynamic yeast populations were evaluated. 2. Materials and methods 2.1. Cider fermentations The present study has been carried out in one cellar from Villaviciosa (Asturias). Asturias is a cold region placed in the north of Spain. Two cider-making systems (traditional and alternative) were studied in this cellar, where commercial dry yeast had never been used. In the traditional system, the must was obtained by means of a batch mechanical press with a slow pressing cycle (3 days), the fermentation process being conducted in a 10,000 -l wood barrel; in the other system, the must was obtained by a pneumatics press (pressing-cycle, 8 h), and the fermentation was carried out in a 25,000-l stainless-steel tank. The study was carried out in the harvests 2001 and 2002. The apple juices (density: 1.050–1.047 mg/l) were obtained from a mixture of acidic, bitter and sweet apples, spontaneously fermented at 12–15 1C (cellar temperature), without SO2 adding. The rates of alcoholic fermentations were monitored by density measuring. 2.2. Yeast sampling and isolation A total of four samples were taken from each vat at different stages of cider production: must (density: 1.050–1.047 mg/l; day 1), beginning of fermentation (density: 1.037–1.020 mg/l; day 4), tumultuous fermentation (density: 1.020–1.010 mg/l) and end of fermentation (density p1.000 mg/l). At each stage, aliquots of several dilutions were spread onto Malt Extract Agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, 2% agar) supplemented with 25 mg/l penicillin G potassium salt (Calbiochem, Nottingham, UK) and 100 mg/l streptomycin sulphate (Acofarma, Madrid, Spain) to inhibit bacterial growth. Plates were incubated for colony development at 28 1C for 2 days. Fifty colonies from each of these samples were randomly picked up. The isolated yeasts were preserved onto Yeast Extract Peptone Dextrose agar slants and stored at 4 1C.
2.3. Molecular identification of yeasts Yeast colonies were assigned to the species level by amplification and restriction of rRNA gene region (Esteve-Zarzoso et al., 1999). For PCR the primers ITS1 (50 -CCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) were used to amplify two variable regions on the 5.8S rDNA gene (White et al., 1990). Cells were collected from agar slant with a sterile toothpick and resuspended in 70 ml of PCR mixture containing 50 mM ITS1, 50 mM ITS4, 0.25 mM dNTPs (Bioline Ltd, London, UK), 1X NH+ 4 , 2.5 mM MgCl2 and 1.2 U Taq polimerasa BiotaqTM (Bioline Ltd, London, UK). The rRNA gene region was amplified in a thermocycler Gene Amps PCR System 9700 (Perkin–Elmer, Wellesley, MA, USA) under the following conditions: initial denaturing at 94 1C for 5 min; 35 cycles of the denaturing at 94 1C for 1 min, annealing at 55 1C for 1 min, and extension at 72 1C for 2 min; and a final extension step of 10 min at 72 1C. PCR products (12 ml) were digested without further purification with the restriction endonucleases Cfo I, Hae III and Hinf I (Roche, Mannheim, Germany) according to the supplier’s instructions. Eventually others two endonuclease have been used, Dde I to differentiate between the species Hanseniaspora guilliermondii and Hanseniaspora uvarum and Hpa II to differentiate the species S. cerevisiae and S. paradoxus. Amplified products and their restriction fragments were, respectively, electrophoresed on 1.4% and 3% agarose gels, in 1X TAE (Tris–acetic acid-EDTA) buffer at 100 V using a Power-Pac 300 (Bio-Rad, Hercules, CA, USA). Gels were stained with ethidium bromide and DNA fragments were subsequently visualized under UV light and scanned using a camera CCD Gene Genius (Syngene, Cambridge, UK). The restriction fragment sizes were measured as base pairs calculated in comparison with a commercial standard (100-base pairs DNA ladder; Bio-Rad, Hercules, CA, USA). Yeasts were identified to species level by comparison of the amplified product and their restriction fragments sizes with the sizes described elsewhere (Esteve-Zarzoso et al., 1999; Ferna´ndez-Espinar et al., 2000) for control species. Certified yeast strains of different species obtained from the Spanish Type Culture Collection (CECT) were used as a reference strains in each amplification and restriction reactions. Strains: H. uvarum (CECT 1444), Hanseniaspora valbyensis (CECT 10122), Metschnikowia pulcherrima (CECT 10408), Saccharomyces cerevisiae (CECT 1883) and Saccharomyces bayanus (CECT 1969). 2.4. Analytical procedures Density (mg/l), pH and ethanol (% v/v) were determined following the methodology described in the Official Methods of Analysis of UE (1998).
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Sugars, glycerol and sorbitol were analysed by highperformance liquid chromatography using an HPLC (Waters, Milford, MA, USA) apparatus, equipped with a 510 pump, a Waters 717-Plus automatic injector provided with temperature control and an IR 410 refraction index detector. The system was controlled by the Millenium v.3.1 software. Separation was carried out on a Sugar-Pak I cation-exchange column (Waters, Milford, MA, USA) according to the method optimized by Blanco et al. (1988). Standard solutions (glucose, fructose, sucrose, glycerol and sorbitol) were prepared by dilution of the individual compounds in an ethanol/water mixture (5/95). The ethanol (HPLC quality) was purchased from Panreac (Barcelona, Spain) and the ultra pure water was obtained from a Mili-Q system from Millipore (Milford, MA, USA). Organic acids were determined by HPLC (Waters, Milford, MA, USA), using a diode array detector (DAD 996), at 206 nm. The column employed was a Spherisorb ODS-2 (250 mm 0.46 mm i.d.; 3 mm particle size from Waters), the mobile phase was a 0.01 M potassium dihydrogen phosphate buffer solution of pH ¼ 2.43. Elution was performed at 40 1C, in isocratic mode (flow rate 0.5 ml/min). The samples were microfiltered through a 0.45 mm-cellulose acetate filter and directly injected (10 ml) onto the chromatographic column. Standard solutions (malic acid, lactic acid, acetic acid and succinic acid) were prepared by dilution of the individual compounds in ultra pure water. Major volatile compounds were analysed in a HewlettPackard model 5890 series II (Agilent Technologies, PaloAlto, CA, USA) gas chromatograph equipped with a flame ionization detector (FID) and a FFAP semicapillary column (30 m 0.53 mm i.d.; phase thickness, 1.0 mm) supplied by Tecknokroma (Barcelona, Spain). Injection was carried out in splitless mode (1 min) employing helium as the carrier gas at 10 ml/min. The temperature gradient was as follows: 40 1C isotherm for 5 min, followed by a linear increase of 4 1C/min until 60 1C. This temperature was raised to 220 1C at a rate of 10 1C/min. Injector and detector temperatures were 240 and 275 1C respectively. The microfiltered samples were directly injected into the chromatograph (1 ml). Standard solutions (acetaldehyde,
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ethyl acetate, methanol, 1-propanol, i-butanol, 1-butanol, amyl-alcohols, acetoin, ethyl-lactate, hexanol, 2-phenilethanol) were prepared by dilution of the individual compounds in an ethanol/water mixture (5/95). All the analyses were carried out in triplicate. 2.5. Statistical analysis Variance analysis (ANOVA) were done to test the main effects of the two factors studied (cider making technology and harvest) on the analytical characteristics of ciders by means of the SPSS (v.11.5.1) for windows statistical package. 3. Results and discussions A total of 800 yeast colonies isolated from the experimental samples (400 each year) were analysed. The amplification of the ITS1-ITS4 region (size range 400–850 bp) was sufficient to obtain polymorphisms. PCR amplification products showed a high length variation in this region for the different species: 400 bp for the strains of M. pulcherrima, 625 bp for the Pichia guilliermondii, 750 bp for genus Hanseniaspora and 850 bp for the type strains Saccharomyces sensu stricto (Table 1). The digestion of the PCR products with the enzymes Cfo I, Hae III and Hinf I yielded one to five fragments which ranged in size from 75 to 630 bp for Cfo I, from 80 to 780 bp for Hae III and from 105 to 390 bp to Hinf I. The restriction patterns obtained by using endonucleases Dde I or Hpa II enabled to differentiate the species Hanseniaspora guilliermondii from H. uvarum and S. paradoxus from S. cerevisiae, respectively. The yeast species identified and the frequencies of all their rDNA gene RFLP patterns obtained during the spontaneous fermentation in 2001 and 2002 are indicated in Table 2. A wide a variety of yeast species, including Saccharomyces and non-Saccharomyces, were found throughout the cider-making process (Table 2). The species included in the first group, S. bayanus and S. cerevisiae, are characterized by a strong fermentative metabolism, whereas the
Table 1 Size of the PCR and the restriction fragments of the species identified in this study obtained with five different endonucleases AP (bp)
Size of the restriction fragment (bp) CfoI
400 625 750 750 750 850 850
205, 300, 630, 320, 275, 385, 385,
100, 265, 120 310, 150, 365 365
95 60 105 135, 95, 75
Identification species
Hae III
Hinf I
280, 400, 750 750 460, 500, 320,
200, 320, 250, 350, 390, 365, 365,
100 115, 90
120, 90, 80 220, 145 230, 180, 150
190 300 220, 170, 105 200, 180 360 155 155
AP 5.8S-ITS amplified product size. M:Metschnikowia; P: Pichia; H: Hanseniaspora; S: Saccharomyces.
DdeI
HpaII
300, 180, 95, 90, 85
725, 125
M. pulcherrima P. guillermondii H. valbyensis H. uvarum H. osmophila S. bayanus S. cerevisiae
Number of isolates 2001
2002
22 0 164 7 41 130 36
24 1 8 55 2 162 148
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Table 2 Distribution of yeast species (%) during spontaneous fermentations of four apple musts at different sampling stages Yeast species
Hanseniaspora valbyensis Hanseniaspora uvarum Hanseniaspora osmophila Metschnikowia pulcherrima Pichia guillermondii Sacchamomyces bayanus Saccharomyces cerevisiae
2001
2002
Pneumatic pressing
Traditional pressing
Pneumatic pressing
Traditional pressing
Sampling day
Sampling day
Sampling day
Sampling day
1
4
44 12 28 16
2 2
16
28
1
4
16
28
36
36
38
78
94
2 10 82 4
100
8 54
30 14
10 4
12
18 2
38 10
8 2
species belonging to the second group can be classified into two categories: apiculate yeast, with low fermentative activity (H. valbyensis, H. uvarum and Hanseniaspora osmophila), and species mainly showing an oxidative metabolism (M. pulcherrima and Pichia guillermondii). In general, the non-Saccharomyces yeasts grow well during the early stages of fermentation, but are subsequently replaced during the following stages by Saccharomyces yeasts. The presence of these species was described in other surveys of yeast flora in cider factories located in England, France, Ireland and Spain (Beech and Davenport, 1970; Michel et al., 1988; Cabranes, 1994; Morrissey et al., 2004). In order to evaluate the results obtained, the discussion has been divided according to the cider-making technology. 3.1. Alternative cider-making by fast pressing A great population diversity of non-Saccharomyces yeast species was observed in must. In the apple juice, the Hanseniaspora genus represented 84% and 66% of the isolates in 2001 and 2002, respectively (Table 2). During the first year, H. valbyensis (44%) was the major species, followed by H. osmophila, M. pulcherrima and H. uvarum. In the second year, H. uvarum (64%) was the most abundant species, M. pulcherrima was the secondary species and P. guilliermondii and H. osmophila were minor species (2%). From the fourth day (beginning of fermentation) until the end of the fermentation process (density o1.000 mg/l) the Saccharomyces genus was the major one. These results are in agreement with the majority of the ecological surveys of yeast flora of grape and apple must fermentations (Beech and Davenport, 1970; Salih et al., 1988; Cabranes et al., 1990; Martini and Vaughan-Martini, 1990; Fleet and Heard, 1992). Against what was expected, S. bayanus yeasts predominated at the beginning and the tumultuous fermentation steps, while the S. cerevisiae yeasts were the most abundant at the end of the fermentation. However, a number of
6
1
4
7
64 2 32 2
6
4 12
2
4
32 60
70 10
20
26 74
1
4
4 34 2 10
8
2
46 4
48 42
7
28
36 64
28 72
studies showed that the main species in wine fermentation is S. cerevisiae (Querol et al., 1994; Schu¨tz and Gafner, 1993; Sabate et al., 1998; Combina et al., 2004). Nevertheless, the studies done in natural wine strains by genetic and karyotypic analyses showed that S. bayanus is associated with certain types of wines (Tokay, Sauterns), generally found in cold areas (which is the case of Asturias) and defined as cryotolerant species (Castellari and Pacchioli, 1992; Maumov, 1996; Torriani et al., 1999). 3.2. Traditional cider making by slow pressing Contrary to that previously described, in this cidermaking procedure the Saccharomyces genus represented 20% and 50% of the yeasts isolated from fresh must in 2001 and 2002 harvests, respectively (Table 2). With respect to the non-Saccharomyces yeasts, the Hanseniaspora genus represented the 66% and 40% of the isolates in 2001 and 2002, respectively. During the first year, the isolated species were H. valbyensis (36%), H. osmophila (30%) and M. pulcherrima (14%), while in the 2002 harvest the main species was H. uvarum (34%) followed by M. pulcherrima (10%), and minor species (o5%) such as H. valbyensis and H. osmophila. On the other hand, S. bayanus represented in must 18% in 2001 harvest whereas in 2002 this specie rose to 46%. In relation to the source of yeast species, some previous studies showed that Saccharomyces are associated with the surfaces and making equipment of the cellar, thus, its presence is not common in the must, except when there are damaged fruits (Rosini et al., 1982; Martini and Vaughan-Martini, 1990; Gutierrez et al., 1999; Pretorius, 2000). On the contrary, other workers reported the influence of the pressing method on the yeast composition of must (Beech and Carr, 1977; Cuinier, 1980; Poulard et al., 1985). The results obtained in our study were in agreement with these findings, and should be explained because the slow pressing system (3 days) enabled the
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development and growth of the fermentative yeasts coming from the pressing equipment. In the second sample of 2002 (4th day, density: 1.025 mg/l) the species S. bayanus and S. cerevisiae were present at frequencies of 48% and 42%, respectively. At the end of fermentation, these percentages were inverted and a clear majority of S. cerevisiae (72%) was detected. However, in the 2001 harvest an unusual abundance of non-Saccharomyces yeast was found; this fact was due to a notable predominance of the species H. valbyensis during all the steps of fermentation (Table 2). At the beginning of the fermentation (4th day, density: 1.037 mg/l) the Saccharomyces genus increased their frequency to 48% but in the fast fermentation step (16th day, density: 1.019 mg/l) the isolation percentage of this genus decreased until 10%. Simultaneously, in this stage the species H. valbyensis increased its frequency until 78%, reaching 94% at the end of fermentation. The ratio and concentration of the species yeasts present during the fermenting must can be due to several factors, such as geographic location, climatic conditions and making techniques (Querol et al., 1994; Guillamo´n et al., 1998; Gutie´rrez et al., 1999; Sabate et al., 2002). It has been reported that H. valbyensis is usually present in the initial phases of the fermentation of several apple musts (Beech and Davenport, 1970; Michel et al., 1988), but in our study it survived throughout the fermentation process until the end. This fact can be explained by both the cider making technology (fermentation at low temperature, without SO2 added), and the composition of apple musts, with sugar contents lower than 110 g/l, which yields a low alcohol content in the final product. In this sense, it has been reported that apiculate yeasts can tolerate ethanol concentrations higher than 6%
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(Pallman et al., 2001; Combina et al., 2004). These results are in agreement with previous works in ciders (Salih et al., 1988; Cabranes et al., 1990; Sua´rez et al., 2005). 3.3. Analytical characters of the ciders In general, the nature and concentration of volatile and non-volatile compounds in ciders are influenced by: raw material (varieties, ripeness, harvest), the different yeasts that grow during fermentation and the cider making procedures. The analytical composition of the ciders elaborated is shown in Table 3. The alcoholic fermentation was developed independently of the cider-making technology and harvests. The major sugars of the must were metabolized between 20 and 28 days after the apple pressing, and therefore fermentation was always complete. Sorbitol is the main polyalcohol synthesized in the leaves of apple trees and converted into sugars in the fruit. It is not used by yeasts during the fermentation process and contributes to the palate fullness of ciders (Pollard et al., 1966). In apple juice, sorbitol concentration ranged between 6.6 and 6.5 g/l in 2001 and 2002, respectively. As is well known, the glycerol is formed as a result of the metabolism of pyruvic acid during the course of the glycerol-pyruvic fermentation. Fermentation at low temperature increased its concentration (Cabranes et al., 1997; Sua´rez et al., 2005). Levels for glycerol in the ciders analysed varied between 3.6 and 4.8 (Table 3). Non-volatile acids (lactic, acetic, succinic) are important constituents of cider. Malic acid, the main acid in apples, is converted into lactic acid by lactic bacteria. In all the ciders of our study, both the alcoholic and malolactic
Table 3 Analytical characteristics of natural ciders Compound
Density (mg/l) pH Glycerol (g/l) Etanol (% v/v) Sorbitol (g/l) Lactic acid (g/l) Acetic acid (g/l) Succinic acid (g/l) Acetaldehyde (mg/l) Ethyl acetate (mg/l) Methanol (mg/l) 1-Propanol (mg/l) i-Butanol (mg/l) 1-Butanol (mg/l) Amyl alcohols (mg/l) Acetoin (mg/l) Ethyl lactate+hexanol (mg/l) 2-Phenylethanol (mg/l)
Pneumatic pressing
Traditional pressing
2001
2002
2001
2002
1.00170.0001 3.8470.01 4.470.02 6.570.05 6.670.02 4.070.03 0.370.01 0.770.03 370.06 2670.08 4070.58 770.65 2670.05 770.03 19170.23 470.31 15871.99 12170.12
1.00170.0001 3.6770.01 3.670.06 6.670.01 6.570.01 4.670.01 0.270.01 0.570.01 370.03 3370.49 8773.67 1170.15 6770.11 570.05 25370.79 270.14 11070.48 6671.10
0.99970.0001 3.8370.02 4.870.05 6.470.02 6.670.01 4.070.04 0.370.02 0.870.02 270.03 5070.57 4470.41 870.23 2170.21 670.07 14272.17 370.09 10470.85 6870.92
0.99970.0001 3.7770.01 4.470.0001 6.370.03 6.570.01 4.770.02 0.270.01 0.470.04 270.07 2470.30 8273.06 870.18 3570.17 570.01 14770.45 270.12 3770.33 4570.79
Each value in the table represents the mean value7standard deviation from three analyses.
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fermentations were carried out by the indigenous flora and malic acid contents were lower than 0.5 g/l (data not shown). Lactic is the major acid in ciders and succinic acid is the main acid produced by yeasts during the course of the alcoholic and glycerol-pyruvic fermentation, both present at normal levels in Asturian natural ciders (Sua´rez and Picinelli, 2001). With respect to the volatile compounds, methanol and 1butanol were related to the raw material. The results also indicated a low production of acetic acid, acetoin and ethyl acetate, and high amounts of amyl alcohols in all of the ciders. In general, the aromatic profiles are marked by the low ratio of ethyl acetate/higher alcohols (o0.3). In spite of the growth and survival of the H. valbyensis during fermentation of apple must (2001), the final product was not organoleptically affected by its presence (tested by trained experts); the higher concentration of ethyl acetate (50 mg/l) observed in the cider made by the traditional system in 2001 could be explained by the dominance of apiculate yeast in the course of the fermentation. Nevertheless, it is worth to note the high concentrations of 2-phenylethanol found in these natural ciders, according to previous reports on chemical characterization of Asturian cider (Picinelli et al., 2000). This result could be related to the predominance during the tumultuous fermentation step of S. bayanus species, which ability to produce elevated amounts of this aromatic alcohol and its acetate ester has been reported (Masneuf et al., 1998; Querol et al., 2003). Two-way analysis of variance was used to obtain more information about differences in the analytical composition of ciders due to cider making technology and harvest. The results showed that the values of the compounds indicated in Table 3 were not significantly influenced by the cider making technology ðP40:05; traditional versus alternative). However, the harvest factor affected the concentration of lactic acid, methanol and succinic acid ðPo0:05Þ and 1-butanol ðPo0:10Þ. Malic acid, the main acid of the apple juices, is converted to lactic acid by lactic acid bacteria during the malolactic transformation. Thus, the concentration of lactic acid in cider is determined by the apple varieties used and their ripening state (Blanco et al., 2002). However, succinic acid is the main carboxylic acid produced by yeast in the course of the glycerol-piruvic fermentation during cider elaboration. The result of the analysis of variance for succinic acid is in agreement with Giudici et al. (1995), who suggested that the capacity to produce glycerol and succinic acid is a hereditary characteristic of the genus Saccharomyces. In fact, a good relationship was found ðr ¼ 0:6Þ between glycerol and succinic acid in the ciders studied. Among the volatile compounds, 1-butanol is included in the primary aroma of apples (Brown et al., 1966), and methanol is not a yeast fermentation product but it is cleaved from pectins; its concentration depends on several factors such as apple variety, ripening state and cider making practices (Jacquin and Tavernier, 1952).
Acknowledgements The authors are indebted to the Government of the Principado of Asturias (Spain), to the Foundation for Science and Technological Research (FICYT PC-04-24) for financial support and the Town Hall of Villaviciosa (Asturias) for a grant (RPB).
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Pallman, C., Brown, J., Olineka, T., Cocolin, L., Mills, D., Bisson, L., 2001. Use of WL medium of profile native flora fermentations. Am. J. Enol. Viticult. 52, 198–203. Picinelli, A., Suarez, B., Moreno, J., Rodriguez, R., Mangas, J.J., 2000. Chemical characterization of asturian Cider. J. Agr. Food Chem. 48, 3997–4002. Poulard, A., Reberteau, R., Rousset, Y., 1985. La micloflore des phases prefermentaires. Rev. Franc- .Oenol. 99, 14–19. Pollard, A., Kieser, M., Beech, F., 1966. Factors influencing of the flavour of cider. J. Appl. Bacteriol. 29, 369–372. Pretorius, I.S., 2000. Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16, 675–729. Querol, A., Barrio, E., Ramo´n, D., 1994. Population dynamics of natural Saccharomyces strains during wine fermentation. Int. J. Food Microbiol. 21, 315–323. Querol, A., Ferna´ndez-Espinar, M.T., del Olmo, M., Barrio, E., 2003. Adaptive evolution of wine yeast. Int. J. Food Microbiol. 86, 3–10. SPSS., 1994. In: Advanced Statistic 6.1. Marija J. Norusis/SPSS Inc. Chicago. Rosini, G., Federici, F., Martini, A., 1982. Yeast flora of grape berries during ripening. Microbiol. Ecol. 8, 83–89. Sabate, J., Cano, J., Querol, A., Guillamon, J.M., 1998. Diversity of Saccharomyces strains in wine fermentations: analysis for two consecutive years. Lett. Appl. Microbiol. 26, 452–455. Sabate, J., Cano, J., Esteve-Zarzoso, B., Guillamon, J.M., 2002. Isolation and identification of yeasts associated with vineyard and winery by RFPL analysis of ribosomal genes and mitochondrial DNA. Microbiol. Res. 157, 267–274. Salih, A.G., Drilleau, J.F., Cavin, F.F., Divies, C., Bourgeois, C.M., 1988. A survey of microbiological aspects of cider making. J. Inst. Brew. 94, 5–8. Schu¨tz, M., Gafner, J., 1993. Analysis of yeast diversity during spontaneous and induced alcoholic fermentation. J. Appl. Bacteriol. 75, 551–558. Sua´rez, B., Picinelli, A., 2001. Research and development in the cider area in Asturias. Background and perspectives. Recent Res. Devel. Agr. Food Chem. 5, 1–17. Sua´rez, B., Pando, R., Ferna´ndez, N., Gonza´lez, A., Rodriguez, R., 2005. Analytical differentiation of cider inoculated with yeast (Saccharomyce cerevisiae) isolated from Asturian (Spain) apple juice. Lebens.-Wiss. Technol. 38, 455–461. Torriani, S., Zapparoli, G., Suzzi, G., 1999. Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine. Antonie van Leeuwenhoek 75, 207–215. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, pp. 315–322.
Yeast species associated with the spontaneous fermentation of cider Bele´n Sua´rez Vallesa, Rosa Pando Bedrin˜anaa,, Norman Ferna´ndez Tasco´na, Amparo Querol Simo´nb, Roberto Rodrı´ guez Madreraa a
A´rea de Tecnologı´a de los Alimentos, Servicio Regional de Investigacio´n y Desarrollo Agroalimentario (SERIDA) 33300, Villaviciosa, Asturias, Spain b Departamento de Biotecnologı´a, Instituto de Agroquı´mica y Tecnologı´a de los Alimentos (CSIC) 46100, Valencia, Spain Received 1 February 2006; received in revised form 27 March 2006; accepted 1 April 2006 Available online 21 April 2006
Abstract This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by nonSaccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cider yeasts; Spontaneous fermentation; PCR-RFLP
1. Introduction The fermentation of apple must is a complex microbial reaction involving the sequential development of various species of yeasts and bacteria (Beech, 1972; Salih et al., 1988; Cabranes et al., 1990; Duen˜as et al., 1994). Among these micro-organisms, yeasts are primarily responsible for alcoholic fermentation. Thus, the different yeast species developed during fermentation and their dynamics and frequency of appearance determine the taste and flavour characteristics of products (Beech and Davenport, 1970; Mafart, 1986; Le Quere´ and Drilleau, 1993; Cabranes et al., 1997; Sua´rez et al., 2005). In Asturias (Spain), natural cider is produced by the spontaneous fermentation of apple juice by yeasts originated from fruit and cider making equipment. This type of fermentation is of particular interest to ascertain the yeast Corresponding author. Tel.: 34 985 89 00 66; fax: 34 985 891 854.
E-mail address: [email protected] (R.P. Bedrin˜ana). 0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2006.04.001
species associated with the fermentation processes. Preliminary studies about the population dynamic have shown that the genus Saccharomyces is usually predominant during alcoholic fermentation, while the non-Saccharomyces genera, such as Kloeckera, Candida, Pichia, Hansenula, Hanseniaspora and Metschnikowia mainly grow during the first stages of the process (Michel et al., 1988; Cabranes, 1994). On the other hand, several factors such as geographic location, climatic conditions, apple varieties and the cider making technology can influence the diversity of yeasts present in must (Poulard et al., 1985; Cabranes et al., 1990; Mangas et al., 1994; del Campo et al., 2003). Traditionally, the identification and characterization of yeast species have been based on morphological traits and their physiological capabilities (Barnett et al., 1990). The conventional methodology for yeast identification requires evaluation of tests in a complex, laborious and timeconsuming process. In the last decade, microbial identification has undergone a revolutionary change by the introduction of PCR-based methodologies (Ness et al.,
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1993; Paffetti et al., 1995; de Barros Lopes et al., 1996). In this sense, one of the most successful methods for yeast species identification is the restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS) (White et al., 1990; Guillamo´n et al., 1998; Esteve-Zarzoso et al., 1999, 2001; Ferna´ndez-Espinar et al., 2000; Sabate et al., 2002). The aim of this study was to identify the nonSaccharomyces and Saccharomyces biota present in apple musts at different phases of fermentation in one cellar during two consecutive years. Also, the influence of the cider-making systems on the diversity and dynamic yeast populations were evaluated. 2. Materials and methods 2.1. Cider fermentations The present study has been carried out in one cellar from Villaviciosa (Asturias). Asturias is a cold region placed in the north of Spain. Two cider-making systems (traditional and alternative) were studied in this cellar, where commercial dry yeast had never been used. In the traditional system, the must was obtained by means of a batch mechanical press with a slow pressing cycle (3 days), the fermentation process being conducted in a 10,000 -l wood barrel; in the other system, the must was obtained by a pneumatics press (pressing-cycle, 8 h), and the fermentation was carried out in a 25,000-l stainless-steel tank. The study was carried out in the harvests 2001 and 2002. The apple juices (density: 1.050–1.047 mg/l) were obtained from a mixture of acidic, bitter and sweet apples, spontaneously fermented at 12–15 1C (cellar temperature), without SO2 adding. The rates of alcoholic fermentations were monitored by density measuring. 2.2. Yeast sampling and isolation A total of four samples were taken from each vat at different stages of cider production: must (density: 1.050–1.047 mg/l; day 1), beginning of fermentation (density: 1.037–1.020 mg/l; day 4), tumultuous fermentation (density: 1.020–1.010 mg/l) and end of fermentation (density p1.000 mg/l). At each stage, aliquots of several dilutions were spread onto Malt Extract Agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, 2% agar) supplemented with 25 mg/l penicillin G potassium salt (Calbiochem, Nottingham, UK) and 100 mg/l streptomycin sulphate (Acofarma, Madrid, Spain) to inhibit bacterial growth. Plates were incubated for colony development at 28 1C for 2 days. Fifty colonies from each of these samples were randomly picked up. The isolated yeasts were preserved onto Yeast Extract Peptone Dextrose agar slants and stored at 4 1C.
2.3. Molecular identification of yeasts Yeast colonies were assigned to the species level by amplification and restriction of rRNA gene region (Esteve-Zarzoso et al., 1999). For PCR the primers ITS1 (50 -CCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) were used to amplify two variable regions on the 5.8S rDNA gene (White et al., 1990). Cells were collected from agar slant with a sterile toothpick and resuspended in 70 ml of PCR mixture containing 50 mM ITS1, 50 mM ITS4, 0.25 mM dNTPs (Bioline Ltd, London, UK), 1X NH+ 4 , 2.5 mM MgCl2 and 1.2 U Taq polimerasa BiotaqTM (Bioline Ltd, London, UK). The rRNA gene region was amplified in a thermocycler Gene Amps PCR System 9700 (Perkin–Elmer, Wellesley, MA, USA) under the following conditions: initial denaturing at 94 1C for 5 min; 35 cycles of the denaturing at 94 1C for 1 min, annealing at 55 1C for 1 min, and extension at 72 1C for 2 min; and a final extension step of 10 min at 72 1C. PCR products (12 ml) were digested without further purification with the restriction endonucleases Cfo I, Hae III and Hinf I (Roche, Mannheim, Germany) according to the supplier’s instructions. Eventually others two endonuclease have been used, Dde I to differentiate between the species Hanseniaspora guilliermondii and Hanseniaspora uvarum and Hpa II to differentiate the species S. cerevisiae and S. paradoxus. Amplified products and their restriction fragments were, respectively, electrophoresed on 1.4% and 3% agarose gels, in 1X TAE (Tris–acetic acid-EDTA) buffer at 100 V using a Power-Pac 300 (Bio-Rad, Hercules, CA, USA). Gels were stained with ethidium bromide and DNA fragments were subsequently visualized under UV light and scanned using a camera CCD Gene Genius (Syngene, Cambridge, UK). The restriction fragment sizes were measured as base pairs calculated in comparison with a commercial standard (100-base pairs DNA ladder; Bio-Rad, Hercules, CA, USA). Yeasts were identified to species level by comparison of the amplified product and their restriction fragments sizes with the sizes described elsewhere (Esteve-Zarzoso et al., 1999; Ferna´ndez-Espinar et al., 2000) for control species. Certified yeast strains of different species obtained from the Spanish Type Culture Collection (CECT) were used as a reference strains in each amplification and restriction reactions. Strains: H. uvarum (CECT 1444), Hanseniaspora valbyensis (CECT 10122), Metschnikowia pulcherrima (CECT 10408), Saccharomyces cerevisiae (CECT 1883) and Saccharomyces bayanus (CECT 1969). 2.4. Analytical procedures Density (mg/l), pH and ethanol (% v/v) were determined following the methodology described in the Official Methods of Analysis of UE (1998).
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Sugars, glycerol and sorbitol were analysed by highperformance liquid chromatography using an HPLC (Waters, Milford, MA, USA) apparatus, equipped with a 510 pump, a Waters 717-Plus automatic injector provided with temperature control and an IR 410 refraction index detector. The system was controlled by the Millenium v.3.1 software. Separation was carried out on a Sugar-Pak I cation-exchange column (Waters, Milford, MA, USA) according to the method optimized by Blanco et al. (1988). Standard solutions (glucose, fructose, sucrose, glycerol and sorbitol) were prepared by dilution of the individual compounds in an ethanol/water mixture (5/95). The ethanol (HPLC quality) was purchased from Panreac (Barcelona, Spain) and the ultra pure water was obtained from a Mili-Q system from Millipore (Milford, MA, USA). Organic acids were determined by HPLC (Waters, Milford, MA, USA), using a diode array detector (DAD 996), at 206 nm. The column employed was a Spherisorb ODS-2 (250 mm 0.46 mm i.d.; 3 mm particle size from Waters), the mobile phase was a 0.01 M potassium dihydrogen phosphate buffer solution of pH ¼ 2.43. Elution was performed at 40 1C, in isocratic mode (flow rate 0.5 ml/min). The samples were microfiltered through a 0.45 mm-cellulose acetate filter and directly injected (10 ml) onto the chromatographic column. Standard solutions (malic acid, lactic acid, acetic acid and succinic acid) were prepared by dilution of the individual compounds in ultra pure water. Major volatile compounds were analysed in a HewlettPackard model 5890 series II (Agilent Technologies, PaloAlto, CA, USA) gas chromatograph equipped with a flame ionization detector (FID) and a FFAP semicapillary column (30 m 0.53 mm i.d.; phase thickness, 1.0 mm) supplied by Tecknokroma (Barcelona, Spain). Injection was carried out in splitless mode (1 min) employing helium as the carrier gas at 10 ml/min. The temperature gradient was as follows: 40 1C isotherm for 5 min, followed by a linear increase of 4 1C/min until 60 1C. This temperature was raised to 220 1C at a rate of 10 1C/min. Injector and detector temperatures were 240 and 275 1C respectively. The microfiltered samples were directly injected into the chromatograph (1 ml). Standard solutions (acetaldehyde,
27
ethyl acetate, methanol, 1-propanol, i-butanol, 1-butanol, amyl-alcohols, acetoin, ethyl-lactate, hexanol, 2-phenilethanol) were prepared by dilution of the individual compounds in an ethanol/water mixture (5/95). All the analyses were carried out in triplicate. 2.5. Statistical analysis Variance analysis (ANOVA) were done to test the main effects of the two factors studied (cider making technology and harvest) on the analytical characteristics of ciders by means of the SPSS (v.11.5.1) for windows statistical package. 3. Results and discussions A total of 800 yeast colonies isolated from the experimental samples (400 each year) were analysed. The amplification of the ITS1-ITS4 region (size range 400–850 bp) was sufficient to obtain polymorphisms. PCR amplification products showed a high length variation in this region for the different species: 400 bp for the strains of M. pulcherrima, 625 bp for the Pichia guilliermondii, 750 bp for genus Hanseniaspora and 850 bp for the type strains Saccharomyces sensu stricto (Table 1). The digestion of the PCR products with the enzymes Cfo I, Hae III and Hinf I yielded one to five fragments which ranged in size from 75 to 630 bp for Cfo I, from 80 to 780 bp for Hae III and from 105 to 390 bp to Hinf I. The restriction patterns obtained by using endonucleases Dde I or Hpa II enabled to differentiate the species Hanseniaspora guilliermondii from H. uvarum and S. paradoxus from S. cerevisiae, respectively. The yeast species identified and the frequencies of all their rDNA gene RFLP patterns obtained during the spontaneous fermentation in 2001 and 2002 are indicated in Table 2. A wide a variety of yeast species, including Saccharomyces and non-Saccharomyces, were found throughout the cider-making process (Table 2). The species included in the first group, S. bayanus and S. cerevisiae, are characterized by a strong fermentative metabolism, whereas the
Table 1 Size of the PCR and the restriction fragments of the species identified in this study obtained with five different endonucleases AP (bp)
Size of the restriction fragment (bp) CfoI
400 625 750 750 750 850 850
205, 300, 630, 320, 275, 385, 385,
100, 265, 120 310, 150, 365 365
95 60 105 135, 95, 75
Identification species
Hae III
Hinf I
280, 400, 750 750 460, 500, 320,
200, 320, 250, 350, 390, 365, 365,
100 115, 90
120, 90, 80 220, 145 230, 180, 150
190 300 220, 170, 105 200, 180 360 155 155
AP 5.8S-ITS amplified product size. M:Metschnikowia; P: Pichia; H: Hanseniaspora; S: Saccharomyces.
DdeI
HpaII
300, 180, 95, 90, 85
725, 125
M. pulcherrima P. guillermondii H. valbyensis H. uvarum H. osmophila S. bayanus S. cerevisiae
Number of isolates 2001
2002
22 0 164 7 41 130 36
24 1 8 55 2 162 148
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Table 2 Distribution of yeast species (%) during spontaneous fermentations of four apple musts at different sampling stages Yeast species
Hanseniaspora valbyensis Hanseniaspora uvarum Hanseniaspora osmophila Metschnikowia pulcherrima Pichia guillermondii Sacchamomyces bayanus Saccharomyces cerevisiae
2001
2002
Pneumatic pressing
Traditional pressing
Pneumatic pressing
Traditional pressing
Sampling day
Sampling day
Sampling day
Sampling day
1
4
44 12 28 16
2 2
16
28
1
4
16
28
36
36
38
78
94
2 10 82 4
100
8 54
30 14
10 4
12
18 2
38 10
8 2
species belonging to the second group can be classified into two categories: apiculate yeast, with low fermentative activity (H. valbyensis, H. uvarum and Hanseniaspora osmophila), and species mainly showing an oxidative metabolism (M. pulcherrima and Pichia guillermondii). In general, the non-Saccharomyces yeasts grow well during the early stages of fermentation, but are subsequently replaced during the following stages by Saccharomyces yeasts. The presence of these species was described in other surveys of yeast flora in cider factories located in England, France, Ireland and Spain (Beech and Davenport, 1970; Michel et al., 1988; Cabranes, 1994; Morrissey et al., 2004). In order to evaluate the results obtained, the discussion has been divided according to the cider-making technology. 3.1. Alternative cider-making by fast pressing A great population diversity of non-Saccharomyces yeast species was observed in must. In the apple juice, the Hanseniaspora genus represented 84% and 66% of the isolates in 2001 and 2002, respectively (Table 2). During the first year, H. valbyensis (44%) was the major species, followed by H. osmophila, M. pulcherrima and H. uvarum. In the second year, H. uvarum (64%) was the most abundant species, M. pulcherrima was the secondary species and P. guilliermondii and H. osmophila were minor species (2%). From the fourth day (beginning of fermentation) until the end of the fermentation process (density o1.000 mg/l) the Saccharomyces genus was the major one. These results are in agreement with the majority of the ecological surveys of yeast flora of grape and apple must fermentations (Beech and Davenport, 1970; Salih et al., 1988; Cabranes et al., 1990; Martini and Vaughan-Martini, 1990; Fleet and Heard, 1992). Against what was expected, S. bayanus yeasts predominated at the beginning and the tumultuous fermentation steps, while the S. cerevisiae yeasts were the most abundant at the end of the fermentation. However, a number of
6
1
4
7
64 2 32 2
6
4 12
2
4
32 60
70 10
20
26 74
1
4
4 34 2 10
8
2
46 4
48 42
7
28
36 64
28 72
studies showed that the main species in wine fermentation is S. cerevisiae (Querol et al., 1994; Schu¨tz and Gafner, 1993; Sabate et al., 1998; Combina et al., 2004). Nevertheless, the studies done in natural wine strains by genetic and karyotypic analyses showed that S. bayanus is associated with certain types of wines (Tokay, Sauterns), generally found in cold areas (which is the case of Asturias) and defined as cryotolerant species (Castellari and Pacchioli, 1992; Maumov, 1996; Torriani et al., 1999). 3.2. Traditional cider making by slow pressing Contrary to that previously described, in this cidermaking procedure the Saccharomyces genus represented 20% and 50% of the yeasts isolated from fresh must in 2001 and 2002 harvests, respectively (Table 2). With respect to the non-Saccharomyces yeasts, the Hanseniaspora genus represented the 66% and 40% of the isolates in 2001 and 2002, respectively. During the first year, the isolated species were H. valbyensis (36%), H. osmophila (30%) and M. pulcherrima (14%), while in the 2002 harvest the main species was H. uvarum (34%) followed by M. pulcherrima (10%), and minor species (o5%) such as H. valbyensis and H. osmophila. On the other hand, S. bayanus represented in must 18% in 2001 harvest whereas in 2002 this specie rose to 46%. In relation to the source of yeast species, some previous studies showed that Saccharomyces are associated with the surfaces and making equipment of the cellar, thus, its presence is not common in the must, except when there are damaged fruits (Rosini et al., 1982; Martini and Vaughan-Martini, 1990; Gutierrez et al., 1999; Pretorius, 2000). On the contrary, other workers reported the influence of the pressing method on the yeast composition of must (Beech and Carr, 1977; Cuinier, 1980; Poulard et al., 1985). The results obtained in our study were in agreement with these findings, and should be explained because the slow pressing system (3 days) enabled the
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development and growth of the fermentative yeasts coming from the pressing equipment. In the second sample of 2002 (4th day, density: 1.025 mg/l) the species S. bayanus and S. cerevisiae were present at frequencies of 48% and 42%, respectively. At the end of fermentation, these percentages were inverted and a clear majority of S. cerevisiae (72%) was detected. However, in the 2001 harvest an unusual abundance of non-Saccharomyces yeast was found; this fact was due to a notable predominance of the species H. valbyensis during all the steps of fermentation (Table 2). At the beginning of the fermentation (4th day, density: 1.037 mg/l) the Saccharomyces genus increased their frequency to 48% but in the fast fermentation step (16th day, density: 1.019 mg/l) the isolation percentage of this genus decreased until 10%. Simultaneously, in this stage the species H. valbyensis increased its frequency until 78%, reaching 94% at the end of fermentation. The ratio and concentration of the species yeasts present during the fermenting must can be due to several factors, such as geographic location, climatic conditions and making techniques (Querol et al., 1994; Guillamo´n et al., 1998; Gutie´rrez et al., 1999; Sabate et al., 2002). It has been reported that H. valbyensis is usually present in the initial phases of the fermentation of several apple musts (Beech and Davenport, 1970; Michel et al., 1988), but in our study it survived throughout the fermentation process until the end. This fact can be explained by both the cider making technology (fermentation at low temperature, without SO2 added), and the composition of apple musts, with sugar contents lower than 110 g/l, which yields a low alcohol content in the final product. In this sense, it has been reported that apiculate yeasts can tolerate ethanol concentrations higher than 6%
29
(Pallman et al., 2001; Combina et al., 2004). These results are in agreement with previous works in ciders (Salih et al., 1988; Cabranes et al., 1990; Sua´rez et al., 2005). 3.3. Analytical characters of the ciders In general, the nature and concentration of volatile and non-volatile compounds in ciders are influenced by: raw material (varieties, ripeness, harvest), the different yeasts that grow during fermentation and the cider making procedures. The analytical composition of the ciders elaborated is shown in Table 3. The alcoholic fermentation was developed independently of the cider-making technology and harvests. The major sugars of the must were metabolized between 20 and 28 days after the apple pressing, and therefore fermentation was always complete. Sorbitol is the main polyalcohol synthesized in the leaves of apple trees and converted into sugars in the fruit. It is not used by yeasts during the fermentation process and contributes to the palate fullness of ciders (Pollard et al., 1966). In apple juice, sorbitol concentration ranged between 6.6 and 6.5 g/l in 2001 and 2002, respectively. As is well known, the glycerol is formed as a result of the metabolism of pyruvic acid during the course of the glycerol-pyruvic fermentation. Fermentation at low temperature increased its concentration (Cabranes et al., 1997; Sua´rez et al., 2005). Levels for glycerol in the ciders analysed varied between 3.6 and 4.8 (Table 3). Non-volatile acids (lactic, acetic, succinic) are important constituents of cider. Malic acid, the main acid in apples, is converted into lactic acid by lactic bacteria. In all the ciders of our study, both the alcoholic and malolactic
Table 3 Analytical characteristics of natural ciders Compound
Density (mg/l) pH Glycerol (g/l) Etanol (% v/v) Sorbitol (g/l) Lactic acid (g/l) Acetic acid (g/l) Succinic acid (g/l) Acetaldehyde (mg/l) Ethyl acetate (mg/l) Methanol (mg/l) 1-Propanol (mg/l) i-Butanol (mg/l) 1-Butanol (mg/l) Amyl alcohols (mg/l) Acetoin (mg/l) Ethyl lactate+hexanol (mg/l) 2-Phenylethanol (mg/l)
Pneumatic pressing
Traditional pressing
2001
2002
2001
2002
1.00170.0001 3.8470.01 4.470.02 6.570.05 6.670.02 4.070.03 0.370.01 0.770.03 370.06 2670.08 4070.58 770.65 2670.05 770.03 19170.23 470.31 15871.99 12170.12
1.00170.0001 3.6770.01 3.670.06 6.670.01 6.570.01 4.670.01 0.270.01 0.570.01 370.03 3370.49 8773.67 1170.15 6770.11 570.05 25370.79 270.14 11070.48 6671.10
0.99970.0001 3.8370.02 4.870.05 6.470.02 6.670.01 4.070.04 0.370.02 0.870.02 270.03 5070.57 4470.41 870.23 2170.21 670.07 14272.17 370.09 10470.85 6870.92
0.99970.0001 3.7770.01 4.470.0001 6.370.03 6.570.01 4.770.02 0.270.01 0.470.04 270.07 2470.30 8273.06 870.18 3570.17 570.01 14770.45 270.12 3770.33 4570.79
Each value in the table represents the mean value7standard deviation from three analyses.
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fermentations were carried out by the indigenous flora and malic acid contents were lower than 0.5 g/l (data not shown). Lactic is the major acid in ciders and succinic acid is the main acid produced by yeasts during the course of the alcoholic and glycerol-pyruvic fermentation, both present at normal levels in Asturian natural ciders (Sua´rez and Picinelli, 2001). With respect to the volatile compounds, methanol and 1butanol were related to the raw material. The results also indicated a low production of acetic acid, acetoin and ethyl acetate, and high amounts of amyl alcohols in all of the ciders. In general, the aromatic profiles are marked by the low ratio of ethyl acetate/higher alcohols (o0.3). In spite of the growth and survival of the H. valbyensis during fermentation of apple must (2001), the final product was not organoleptically affected by its presence (tested by trained experts); the higher concentration of ethyl acetate (50 mg/l) observed in the cider made by the traditional system in 2001 could be explained by the dominance of apiculate yeast in the course of the fermentation. Nevertheless, it is worth to note the high concentrations of 2-phenylethanol found in these natural ciders, according to previous reports on chemical characterization of Asturian cider (Picinelli et al., 2000). This result could be related to the predominance during the tumultuous fermentation step of S. bayanus species, which ability to produce elevated amounts of this aromatic alcohol and its acetate ester has been reported (Masneuf et al., 1998; Querol et al., 2003). Two-way analysis of variance was used to obtain more information about differences in the analytical composition of ciders due to cider making technology and harvest. The results showed that the values of the compounds indicated in Table 3 were not significantly influenced by the cider making technology ðP40:05; traditional versus alternative). However, the harvest factor affected the concentration of lactic acid, methanol and succinic acid ðPo0:05Þ and 1-butanol ðPo0:10Þ. Malic acid, the main acid of the apple juices, is converted to lactic acid by lactic acid bacteria during the malolactic transformation. Thus, the concentration of lactic acid in cider is determined by the apple varieties used and their ripening state (Blanco et al., 2002). However, succinic acid is the main carboxylic acid produced by yeast in the course of the glycerol-piruvic fermentation during cider elaboration. The result of the analysis of variance for succinic acid is in agreement with Giudici et al. (1995), who suggested that the capacity to produce glycerol and succinic acid is a hereditary characteristic of the genus Saccharomyces. In fact, a good relationship was found ðr ¼ 0:6Þ between glycerol and succinic acid in the ciders studied. Among the volatile compounds, 1-butanol is included in the primary aroma of apples (Brown et al., 1966), and methanol is not a yeast fermentation product but it is cleaved from pectins; its concentration depends on several factors such as apple variety, ripening state and cider making practices (Jacquin and Tavernier, 1952).
Acknowledgements The authors are indebted to the Government of the Principado of Asturias (Spain), to the Foundation for Science and Technological Research (FICYT PC-04-24) for financial support and the Town Hall of Villaviciosa (Asturias) for a grant (RPB).
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