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Impact of mother sediment on yeast growth, biodiversity, and ethanol production during fermentation of Vinsanto wine
International Journal of Food Microbiology 129 (2009) 83–87
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Impact of mother sediment on yeast growth, biodiversity, and ethanol production during fermentation of Vinsanto wine Paola Domizio a,⁎, Ilaria Manazzu b, Maurizio Ciani c a b c
Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Via Donizetti 6, 50144 Firenze, Italy Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agro-Alimentari, Università degli Studi di Sassari,Viale Italia 39, 07100 Sassari, Italy Dipartimento S.A.I.F.E.T. sez. di Microbiologia Alimentare, Industriale e Ambientale, Università Politecnica delle Marche,Via Brecce Bianche, 60131 Ancona, Italy
a r t i c l e
i n f o
Article history: Received 7 July 2008 Received in revised form 1 October 2008 Accepted 14 October 2008 Keywords: Mother sediment Yeast biodiversity Ethanol production Vinsanto wine
a b s t r a c t The aim of this study was to determine the impact of Vinsanto mother sediment both on the growth and biodiversity of the yeast microflora and on the production of ethanol under natural and inoculated fermentation of Vinsanto wines. To achieve this ten fermentation trials were carried out in 50-L barrels, five without added mother sediment and five with. Moreover, eight of the ten barrels were inoculated with four Saccharomyces cerevisiae wine strains, while the remaining two barrels were not inoculated and were used as controls to study the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The counts of viable yeasts at three different sampling times indicated that the mother sediment had a positive influence on yeast growth and persistence during fermentation. Molecular characterization of the Saccharomyces type colonies isolated after three months of fermentation showed that the addition of mother sediment had no effects on the dominance of the wine starters. In contrast, the mother sediment had a positive influence on the biodiversity of the spontaneous S. cerevisiae yeasts. Moreover, possibly due to its content of fatty acids and sterols and other nutrients, the addition of mother sediment also showed a positive effect on the fermentative activities of wine yeasts as measured by their ethanol production. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Vinsanto is a dessert wine that is characteristic of Tuscany and some other viticultural areas of central and northern Italy. The traditional production of Vinsanto is based on the fermentation and biological ageing of a must that is obtained from grapes that have been partially dried for 3 to 4 months. The resulting high-sugar-containing must is used to fill 50 L to 200 L wooden barrels known as caratelli, and added with 5% to 10% of the sediment collected from the barrels of the previous Vinsanto wine production, known as the mother sediment. The mother sediment contains the residues of lees, gums and other insoluble high-molecular-weight substances (Tachis, 1968). Thus, according to Gómez et al. (2004) who analyzed the composition of lees from Sherry wines, mother sediment can be a source of lipids that are useful for yeast growth under fermentative conditions. According to the traditional production process, the fermentation occurs in a special room known as the vinsantaia, under ambient temperatures and humidity. The dramatic variations in environmental temperatures through the whole fermentation process strongly influence the growth and fermentative abilities of the wine yeasts. Previous work (Domizio et al., 2007) indicated that the low temperatures that ⁎ Corresponding author. Tel.: +39 055 3220328; fax: +39 055 355995. E-mail address: domizio@unifi.it (P. Domizio). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.10.016
occur during the initial stages of fermentation exert different effects on the wine yeasts. On the one hand, they can have a negative influence on the growth of spontaneous or inoculated S. cerevisiae strains, which will reach a peak in cell concentration only when the temperature starts to get warmer. On the other hand, low temperatures decrease the ethanol sensitivity of non-Saccharomyces yeasts, thus allowing their prolonged survival during fermentation. Vinsanto fermentation and ageing require two years or more, and they result in demi-sec or lightly sweet wines that are characterized by a yellow-amber colour and high alcohol and glycerol contents (Stella et al., 1998). Due to poor management of technological parameters, such as the temperature of fermentation and ageing and the size and biodiversity of the microbial population involved in the fermentation, the sensorial properties of Vinsanto vary from year to year and from one caratello to another. To standardise their process and to obtain Vinsanto wines with valuable and reproducible characteristics, some wineries inoculate the must with commercial yeast strains and carry out the fermentation under controlled temperatures. Another variable that can affect the final quality of Vinsanto is the mother sediment. According to popular tradition, mother sediment is a source of naturally selected fermenting yeasts. However, a recent study reported on the presence of Zygosaccharomyces yeasts in mother sediment, and highlighted that it was not possible to isolate yeasts belonging to the genus Saccharomyces from
84
P. Domizio et al. / International Journal of Food Microbiology 129 (2009) 83–87
2. Materials and methods
(Sigma Aldrich Inc., St. Louis, MO, USA) (0.015%) to inhibit the development of moulds. Plates that contained between 30 and 300 colonies were used to calculate yeast populations. Three months after the inoculation, ten Saccharomyces type colonies (based on their colour and morphology) for each trial were selected at random, purified onto Yeast Peptone Dextrose plates (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar), maintained on YPD slants at 4 °C and subjected to molecular characterization.
2.1. Wine production
2.4. DNA extraction and PCR conditions
The fermentation trials were carried out in the vinsantaia of a long established plant (Antinori, Santa Cristina-Tavarnelle, Florence, Italy), according to the traditional manufacture of Vinsanto. Briefly, Trebbiano, Malvasia and Grechetto grape varieties were harvested during the 2005 vintage and dried on special mats in the fruttaio, a room exposed to ambient temperatures and traditionally devoted to grape drying. During drying no evident mould growth was observed on grapes. After three months, the dried grapes were pressed without sulphite addition and a must characterized by a sugar concentration of 35% (w/v) and pH 3.5 was obtained. Ten 50-L barrels were washed and steam sanitized, and then filled with 40 L of must. The fermentation occurred under the vinsantaia conditions (ambient temperatures and moisture). Five barrels had no mother sediment additions and five barrels were added with 10% of mother sediment. Each one of four S. cerevisiae strains was inoculated into two barrels (one without and one with mother sediment), according to the experimental design reported in Table 1. The remaining two barrels were not inoculated and served as controls for the study of the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The addition of mother sediment resulted in the dilution of sugar concentration (to 32.8% w/v) and the addition of ethanol (1.2% v/v) to the must.
PCR reactions were performed on the total genomic DNA extracted from overnight liquid cultures, as described by Ushinsky et al. (1997). Approximately 20 ng of template DNA was used for the PCR reactions. These were performed on a Perkin Elmer Gene AMP PCR System 9700, in 25 μl reaction mixtures. HSP150 and SED1 were amplified as described by Marinangeli et al. (2004). For inter-δ sequence amplification, the template DNAs were added to the PCR reaction mix containing 0.4 U Taq polymerase (Amersham-Biosciences UK Limited, Buckinghamshire, UK), 1× PCR buffer (MgCl2 free), 2.5 mM MgCl2, 200 mM of each DNTP, and 1 μM of each primer. The primers used were δ1 (5′-CAAAATTCACCTATW[A/T]TCTCA-3′) and δ12 (5′TCAACAATGGAATCCCAAC-3′) (Legras and Karst, 2003). The PCR reactions were carried out according to the following programme: heat denaturation at 95 °C for 4 min, followed by 35 cycles (95 °C for 30 s, 46 °C for 30 s and 72 °C for 2 min) and a final elongation step of 2 min at 72 °C. The PCR products were analysed by electrophoresis on 1.4% agarose gels in 1X TBE buffer. The gel images were visualised using a Biorad Gel DOC 1000 and acquired with Multi-Analyst software (BioRad Laboratories, Hercules, CA, USA).
this substrate by means of classical isolation methods, either with or without enrichment (Domizio et al., 2007). Thus the role of mother sediment in the production of Vinsanto is still a matter of debate. In the present study, we have investigated the effects of mother sediment on ethanol production and on the biodiversity of the yeast population during spontaneous and inoculated fermentations.
2.5. Analytical parameters Ethanol, volatile acidity and reducing sugar were quantified according to the Official EU Methods (EC, 2000).
2.2. Yeast strains The S. cerevisiae strains used were: SS1 and SS2 isolated from Vernaccia wine and belonging to the culture collection of the Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari (DISAABA) of the University of Sassari; DBVPG 1545 belonging to the Industrial Yeasts Collection of the Dipartimento di Biologia Vegetale of the University of Perugia (DBVPG), and the Sherry yeast, a commercial strain purchased from Unican Foods Company Limited (Norwich, England) (Table 1). 2.3. Isolation, preliminary identification and maintenance of the yeast isolates Must samples were taken from each barrel immediately after their inoculation (time 0) and three and five months later (times 1 and 2, respectively). One hundred microliters aliquots of serial dilutions of each sample were plated onto both Lysine Agar (LA medium; Oxoid Unipath Ltd, Hampshire, UK), and Wallerstein Laboratory nutrient agar medium (WL medium; Oxoid Unipath Ltd, Hampshire, UK) (Pallman et al., 2001). The latter was supplemented with biphenyl
Table 1 Details of the fermentation trials carried out in the present study Inoculated strain of Saccharomyces cerevisiae
Barrels Without mother sediment
With mother sediment
Strain SS1 (DISAABA) Strain SS2 (DISAABA) Strain 1545 (DBVPG) Strain “Sherry Yeast” (Unican) Control (without inoculum)
F1 F2 F3 F4 F5
F6 F7 F8 F9 F10
3. Results and discussion 3.1. Effects of mother sediment on yeast growth and ethanol production Viable plate counts of the ten barrels were carried out at three sampling times on both WL and LA media (Fig. 1). WL is a differential medium that allows the preliminary identification of wine yeasts on the basis of the colour and morphology of their colonies (Pallman et al., 2001). This medium was therefore used both to evaluate the viable yeast population and the yeast biodiversity in Vinsanto, and to differentiate the Saccharomyces type colonies. LA is a selective medium that does not support the growth of S. cerevisiae (Lin, 1975). The viable plate counts for the non-Saccharomyces population were carried out on this medium. The results obtained immediately after barrel filling (time 0) indicated that the number of Saccharomyces type colonies varied from 1 × 105 to 1.2 × 106 CFU ml− 1 in the inoculated barrels. This variability in viable counts could be due to the high sugar concentration of the must that might have determined different rates of cell death upon inoculation but it could be also a consequence of experimental variability. No Saccharomyces type colonies were detected by viable plate counts in the non-inoculated barrels, with or without the mother sediment. At time 1 (three months after inoculation), in the barrels inoculated with the starter strains and in the presence of the mother sediment, the Saccharomyces type colonies had increased their initial concentrations 5-fold to 30-fold, while an increase of 0.7-fold to 9-fold was seen for the samples without mother sediment (Fig. 1). A similar trend was seen in the non-inoculated barrels F5 and F10 where, in presence of the mother sediment (F10), the number of Saccharomyces type colonies was two orders of magnitude higher than in the corresponding samples without
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Fig. 1. Viable plate counts of Saccharomyces (□) and non-Saccharomyces (○) yeasts from the fermentation trials carried out with and without addition of mother sediment.
mother sediment (F5) (4.5 × 106 CFU ml− 1 and 9 × 104 CFU ml− 1, respectively). Thus, the addition of the mother sediment provided a positive effect on S. cerevisiae growth in natural and inoculated fermentations. At time 2 (five months after inoculation), when a further increase in ethanol concentrations was seen, no more Saccharomyces type colonies were detected on the plates. The non-Saccharomyces yeasts (Fig. 1) were present at concentrations from 8 × 105 CFU ml− 1 to 1.3 × 106 CFU ml− 1 at time 0. After three months (time 1), in spite of the high ethanol concentrations (11.2%– 13.6%, v/v), about 4–7 × 105 CFU ml− 1 of non-Saccharomyces yeasts were still present in the majority of the fermentation trials, while there was a marked decrease in trials F2 and F5 (both without mother sediment) in which 4 × 104 CFU ml− 1 and 1.2 × 105 CFU ml− 1 were counted, respectively. On the basis of morphological features nonSaccharomyces yeasts, mainly ascribed to the Zygosaccharomyces genus, were still present at time 2 in the trials F7, F9 and F10 (all with mother sediment). As already seen in a previous study (Domizio et al., 2007), the mother sediment can serve as a source of these yeasts, and their persistence in Vinsanto wines at time 2 could be explained by their adaptation to the Vinsanto conditions and their well known tolerance to high ethanol and sugar concentrations. The mother sediment also showed a positive effect on fermentative performance, as its presence in the barrels was always accompanied by a significant increase in ethanol production (Table 2). Other studies have shown that wine yeasts can incorporate exogenous lipids under anaerobic conditions (Luparia et al., 2004) and use them to modulate
Table 2 Ethanol, residual sugar and volatile acidity of Vinsanto wines fermented with and without mother sediment at sampling time 2 Barrels
Ethanol (%v/v)
Residual sugar (% w/v)
Volatile acidity (g/l acetic acid)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
13.35 12.38 12.16 12.54 11.96 15.20 15.58 15.15 14.70 13.43
12.63 14.31 14.67 13.75 15.00 10.07 9.07 9.91 10.20 12.64
0.90 0.41 0.61 0.65 0.43 1.35 0.96 1.16 0.90 0.56
the lipid composition of their cell membranes in response to environmental stimuli (Belviso et al., 2004). Thus, the observed effects of the mother sediment on yeast growth and ethanol production could be seen as a consequence of the use of these lipids that are released following yeast autolysis (Pueyo et al., 2000). The trials carried out in the presence of mother sediment showed higher volatile acidities, although within the normal range for this type of wines. This could be due to the persistence of Zygosaccharomyces yeasts, well known producers of acetic acid (Giudici and Zambonelli, 1992). 3.2. Effects of mother sediment on biodiversity of S. cerevisiae At time 1, when about 55% of the total sugar was fermented, ten Saccharomyces type colonies for each inoculated barrel and the S. cerevisiae strains used as fermentation starters were subjected to molecular characterization. For this purpose, PCR amplification was carried out for the HSP150 and SED1 genes (Marinangeli et al., 2004) and the inter-δ sequences (Legras and Karst, 2003), and the molecular profile of each starter was compared with those of the isolates coming from the same barrel. The analyses of the amplification profiles of the target sequences proved to be useful for the separation of the Saccharomyces type colonies and the inoculated starters and the results obtained indicate that the starter Unican Sherry Yeast was the only one to dominate the wild microflora. In particular, seven out of the ten isolates of barrel F4 (without mother sediment) and all of the isolates of barrel F9 (with mother sediment) had the same molecular profile for the starters, with each of the primer pairs used (Fig. 2). Of the remaining strains, only SS1 showed a limited persistence during the fermentation process, while SS2 and DBVPG 1545 could not be reisolated from the inoculated barrels (data not shown). At the same time, ten Saccharomyces type colonies isolated from the two noninoculated barrels (F5 and F10) were subjected to molecular characterization. The results obtained indicated the presence of three and five different biotypes in barrels F5 and F10, without mother sediment and with mother sediment, respectively (Fig. 3). The amplification profiles generated by all of the Saccharomyces type colonies that were isolated from the ten trials and differed from the starter biotypes (from now on indicated as wild Saccharomyces) showed the existence of 10 and 17 different biotypes in barrels without and with mother sediment, respectively (Fig. 3). Interestingly, in barrels F3 and F8, where none of the wild Saccharomyces showed
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P. Domizio et al. / International Journal of Food Microbiology 129 (2009) 83–87
Fig. 2. Molecular characterization of the Saccharomyces type colonies isolated from barrels F4 (without mother sediment) and F9 (with mother sediment) inoculated with starter Unican Sherry yeasts. SY: starter yeast. Lanes 1–10: isolates 1–10. a–bindicate different amplification profiles. L: Gene Ruler™ DNA Ladder Mix (MBI Fermentas); L1O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
the same molecular profile of the starter strain inoculated, a situation similar to that of barrels F5 and F10 was seen. Eight commercial starter strains that are commonly used in wineries were subjected to molecular characterization and compared with those of isolates from the ten barrels. None of the inoculated strains showed the same amplification profile of the wild Saccharomyces. Therefore these last did not seem to derive from cross contamination of the barrels with any of the starter strain used in the winery (Fig. 4). Thus, the biodiversity seen in the natural and inoculated fermentations in the presence of mother sediment might be a consequence of its addition. Domizio et al. (2007) reported that mother sediment does not appear to contain yeasts belonging to the species S. cerevisiae. Thus, the effects of mother sediment on S. cerevisiae growth and biodiversity could be due to its content of lipids and other nutritional factors, which would support mainly the growth of the S. cerevisiae strains naturally present in the must and selected in peculiar environments such as dried grapes, fruttaio and vinsantaia.
Fig. 3. Amplification of inter-δ sequences of the wild Saccharomyces isolated from barrels with and without mother sediment. The numbers given above each lane indicate different strains within each barrel. L1: O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
Interestingly, the addition of mother sediment did not have any effect on the dominance of the inoculated starters. Accordingly, the only strain that dominated the wild microflora (Unican Sherry yeast) did so irrespective of the absence or presence of mother sediment in the barrel. The inability to dominate of the other starters inoculated might be explained considering that fermentation occurs under stressful conditions during Vinsanto winemaking. Indeed, the musts used for the production of Vinsanto had sugar concentrations higher than 26%, and the resulting wines contained high ethanol concentrations at the end of fermentation (≥16%, v/v). In addition, it needs to be noted that the inoculation of the starter strains occurred in February. Thus, at the beginning of fermentation, the yeasts were subjected to low temperatures. These conditions might have negatively affected the dominance of the starter strains that had not been subjected to selective pressure in fermentation processes with similar characteristics. In agreement with this hypothesis the Unican Sherry yeast is normally used for the production of sherry wines in fermentative processes with characteristics similar to those of Vinsanto making. In conclusion, the addition of mother sediment to the must has a positive effect on ethanol production and favours the implantation of wild Saccharomyces and non Saccharomyces strains during fermentation. Since the fermentative activity of wine yeasts with different technological capabilities can improve the complexity of taste and other desirable characteristics of wine (Grossmann et al.,1996; Cheraiti
Fig. 4. Amplification of inter-δ sequences of the eight commercial starter strains (lanes A to H). Asterisks indicate strains that present different amplification profiles with HSP150 and SED1 primers. L1: O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
P. Domizio et al. / International Journal of Food Microbiology 129 (2009) 83–87
et al., 2005, Domizio et al., 2007), the addition of mother sediment to the must can be considered an important practice for the improvement of the final quality of Vinsanto. Acknowledgements The authors wish to thank Marchese Antinori s.r.l., who provided the wine cellar facilities and the financial support for the realization of this study, Marilena Budroni for providing strains SS1 and SS2, and Cristina Bianchi for her valuable contribution to the molecular analyses. References Belviso, S., Bardi, L., Biondi Bartolini, A., Marzona, M., 2004. Lipid nutrition of Saccharomyces cerevisiae in winemaking. Canadian Journal of Microbiology 50, 669–674. Cheraiti, N., Guezenec, S., Salmon, J.M., 2005. Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions. Applied and Environmental Microbiology 71 (1), 255–260. Domizio, P., Lencioni, L., Ciani, M., Di Blasi, S., Pontremolesi, C., Sabatelli, M.P., 2007. Spontaneous and inoculated yeast populations dynamics and their effect on organoleptic characters of Vinsanto wine under different process conditions. International Journal of Food Microbiology 115, 281–289. Giudici, P., Zambonelli, C., 1992. Biometric and genetic study on acetic acid production for breeding of wine yeast. American Journal of Enology and Viticulture 43, 370–374.
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EC, 2000. Community reference methods for the analysis of spirit drinks. Official Journal of the European Community. Brussels. Reg EC No 2870/00. December 19. Gómez, M.E., Igartuburu, J.M., Pando, E., Rodríguez Luis, F., Mourente, G., 2004. Lipid composition of lees from Sherry wine. Journal of Agricultural and Food Chemistry 52, 4791–4794. Grossmann, M., Linsenmeyer, H., Muno, H., Rapp, A., 1996. Use of oligo-strain yeast cultures to increase complexity of wine aroma. Viticulture and Enology Science 51, 175–179. Legras, J.L., Karst, F., 2003. Optimisation of inter-δ analysis for Saccharomyces cerevisiae strain characterisation. FEMS Microbiology Letters 221, 249–255. Lin, Y., 1975. Detection of wild yeasts in the brewery. Efficiency of differential media. Journal of the Institute of Brewing 81, 410–417. Luparia, V., Soubeyrand, V., Berges, T., Julien, A., 2004. Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations. Applied Microbiology and Biotechnology 65, 25–32. Marinangeli, P., Angelozzi, D., Ciani, M., Clementi, F., Mannazzu, I., 2004. Minisatellites in Saccharomyces cerevisiae genes encoding cell wall proteins: a new way towards wine strain characterisation. FEMS Yeast Research 4, 427–435. Pallman, C.L., Brown, J.A., Olineka, T.L., Cocolin, L., Mills, D.A., Bisson, L., 2001. Use of WL medium to profile native flora fermentations. American Journal of Enology and Viticulture 52, 198–203. Pueyo, E., Martínez-Rodríguez, A., Polo, M.C., Santa-María, G., Bartolomé, B., 2000. Release of lipids during yeast autolysis in a model wine system. Journal of Agriculture and Food Chemistry 48, 116–122. Stella, C., Aiello, C., Sabatelli, M.P., 1998. Vinsanto toscano: ricerche per una sua caratterizzazione. Agricoltura Ricerca 173, 81–86. Tachis, G., 1968. Il Libro del Vin Santo. Bonechi, Florence, Italy. Ushinsky, S.C., Bussey, H., Ahmed, A.A., Wang, Y., Friesen, J., Williams, B.A., Storms, R.K., 1997. Histone H1 in Saccharomyces cerevisiae. Yeast 13, 151–161.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Impact of mother sediment on yeast growth, biodiversity, and ethanol production during fermentation of Vinsanto wine Paola Domizio a,⁎, Ilaria Manazzu b, Maurizio Ciani c a b c
Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Via Donizetti 6, 50144 Firenze, Italy Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agro-Alimentari, Università degli Studi di Sassari,Viale Italia 39, 07100 Sassari, Italy Dipartimento S.A.I.F.E.T. sez. di Microbiologia Alimentare, Industriale e Ambientale, Università Politecnica delle Marche,Via Brecce Bianche, 60131 Ancona, Italy
a r t i c l e
i n f o
Article history: Received 7 July 2008 Received in revised form 1 October 2008 Accepted 14 October 2008 Keywords: Mother sediment Yeast biodiversity Ethanol production Vinsanto wine
a b s t r a c t The aim of this study was to determine the impact of Vinsanto mother sediment both on the growth and biodiversity of the yeast microflora and on the production of ethanol under natural and inoculated fermentation of Vinsanto wines. To achieve this ten fermentation trials were carried out in 50-L barrels, five without added mother sediment and five with. Moreover, eight of the ten barrels were inoculated with four Saccharomyces cerevisiae wine strains, while the remaining two barrels were not inoculated and were used as controls to study the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The counts of viable yeasts at three different sampling times indicated that the mother sediment had a positive influence on yeast growth and persistence during fermentation. Molecular characterization of the Saccharomyces type colonies isolated after three months of fermentation showed that the addition of mother sediment had no effects on the dominance of the wine starters. In contrast, the mother sediment had a positive influence on the biodiversity of the spontaneous S. cerevisiae yeasts. Moreover, possibly due to its content of fatty acids and sterols and other nutrients, the addition of mother sediment also showed a positive effect on the fermentative activities of wine yeasts as measured by their ethanol production. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Vinsanto is a dessert wine that is characteristic of Tuscany and some other viticultural areas of central and northern Italy. The traditional production of Vinsanto is based on the fermentation and biological ageing of a must that is obtained from grapes that have been partially dried for 3 to 4 months. The resulting high-sugar-containing must is used to fill 50 L to 200 L wooden barrels known as caratelli, and added with 5% to 10% of the sediment collected from the barrels of the previous Vinsanto wine production, known as the mother sediment. The mother sediment contains the residues of lees, gums and other insoluble high-molecular-weight substances (Tachis, 1968). Thus, according to Gómez et al. (2004) who analyzed the composition of lees from Sherry wines, mother sediment can be a source of lipids that are useful for yeast growth under fermentative conditions. According to the traditional production process, the fermentation occurs in a special room known as the vinsantaia, under ambient temperatures and humidity. The dramatic variations in environmental temperatures through the whole fermentation process strongly influence the growth and fermentative abilities of the wine yeasts. Previous work (Domizio et al., 2007) indicated that the low temperatures that ⁎ Corresponding author. Tel.: +39 055 3220328; fax: +39 055 355995. E-mail address: domizio@unifi.it (P. Domizio). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.10.016
occur during the initial stages of fermentation exert different effects on the wine yeasts. On the one hand, they can have a negative influence on the growth of spontaneous or inoculated S. cerevisiae strains, which will reach a peak in cell concentration only when the temperature starts to get warmer. On the other hand, low temperatures decrease the ethanol sensitivity of non-Saccharomyces yeasts, thus allowing their prolonged survival during fermentation. Vinsanto fermentation and ageing require two years or more, and they result in demi-sec or lightly sweet wines that are characterized by a yellow-amber colour and high alcohol and glycerol contents (Stella et al., 1998). Due to poor management of technological parameters, such as the temperature of fermentation and ageing and the size and biodiversity of the microbial population involved in the fermentation, the sensorial properties of Vinsanto vary from year to year and from one caratello to another. To standardise their process and to obtain Vinsanto wines with valuable and reproducible characteristics, some wineries inoculate the must with commercial yeast strains and carry out the fermentation under controlled temperatures. Another variable that can affect the final quality of Vinsanto is the mother sediment. According to popular tradition, mother sediment is a source of naturally selected fermenting yeasts. However, a recent study reported on the presence of Zygosaccharomyces yeasts in mother sediment, and highlighted that it was not possible to isolate yeasts belonging to the genus Saccharomyces from
84
P. Domizio et al. / International Journal of Food Microbiology 129 (2009) 83–87
2. Materials and methods
(Sigma Aldrich Inc., St. Louis, MO, USA) (0.015%) to inhibit the development of moulds. Plates that contained between 30 and 300 colonies were used to calculate yeast populations. Three months after the inoculation, ten Saccharomyces type colonies (based on their colour and morphology) for each trial were selected at random, purified onto Yeast Peptone Dextrose plates (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar), maintained on YPD slants at 4 °C and subjected to molecular characterization.
2.1. Wine production
2.4. DNA extraction and PCR conditions
The fermentation trials were carried out in the vinsantaia of a long established plant (Antinori, Santa Cristina-Tavarnelle, Florence, Italy), according to the traditional manufacture of Vinsanto. Briefly, Trebbiano, Malvasia and Grechetto grape varieties were harvested during the 2005 vintage and dried on special mats in the fruttaio, a room exposed to ambient temperatures and traditionally devoted to grape drying. During drying no evident mould growth was observed on grapes. After three months, the dried grapes were pressed without sulphite addition and a must characterized by a sugar concentration of 35% (w/v) and pH 3.5 was obtained. Ten 50-L barrels were washed and steam sanitized, and then filled with 40 L of must. The fermentation occurred under the vinsantaia conditions (ambient temperatures and moisture). Five barrels had no mother sediment additions and five barrels were added with 10% of mother sediment. Each one of four S. cerevisiae strains was inoculated into two barrels (one without and one with mother sediment), according to the experimental design reported in Table 1. The remaining two barrels were not inoculated and served as controls for the study of the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The addition of mother sediment resulted in the dilution of sugar concentration (to 32.8% w/v) and the addition of ethanol (1.2% v/v) to the must.
PCR reactions were performed on the total genomic DNA extracted from overnight liquid cultures, as described by Ushinsky et al. (1997). Approximately 20 ng of template DNA was used for the PCR reactions. These were performed on a Perkin Elmer Gene AMP PCR System 9700, in 25 μl reaction mixtures. HSP150 and SED1 were amplified as described by Marinangeli et al. (2004). For inter-δ sequence amplification, the template DNAs were added to the PCR reaction mix containing 0.4 U Taq polymerase (Amersham-Biosciences UK Limited, Buckinghamshire, UK), 1× PCR buffer (MgCl2 free), 2.5 mM MgCl2, 200 mM of each DNTP, and 1 μM of each primer. The primers used were δ1 (5′-CAAAATTCACCTATW[A/T]TCTCA-3′) and δ12 (5′TCAACAATGGAATCCCAAC-3′) (Legras and Karst, 2003). The PCR reactions were carried out according to the following programme: heat denaturation at 95 °C for 4 min, followed by 35 cycles (95 °C for 30 s, 46 °C for 30 s and 72 °C for 2 min) and a final elongation step of 2 min at 72 °C. The PCR products were analysed by electrophoresis on 1.4% agarose gels in 1X TBE buffer. The gel images were visualised using a Biorad Gel DOC 1000 and acquired with Multi-Analyst software (BioRad Laboratories, Hercules, CA, USA).
this substrate by means of classical isolation methods, either with or without enrichment (Domizio et al., 2007). Thus the role of mother sediment in the production of Vinsanto is still a matter of debate. In the present study, we have investigated the effects of mother sediment on ethanol production and on the biodiversity of the yeast population during spontaneous and inoculated fermentations.
2.5. Analytical parameters Ethanol, volatile acidity and reducing sugar were quantified according to the Official EU Methods (EC, 2000).
2.2. Yeast strains The S. cerevisiae strains used were: SS1 and SS2 isolated from Vernaccia wine and belonging to the culture collection of the Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari (DISAABA) of the University of Sassari; DBVPG 1545 belonging to the Industrial Yeasts Collection of the Dipartimento di Biologia Vegetale of the University of Perugia (DBVPG), and the Sherry yeast, a commercial strain purchased from Unican Foods Company Limited (Norwich, England) (Table 1). 2.3. Isolation, preliminary identification and maintenance of the yeast isolates Must samples were taken from each barrel immediately after their inoculation (time 0) and three and five months later (times 1 and 2, respectively). One hundred microliters aliquots of serial dilutions of each sample were plated onto both Lysine Agar (LA medium; Oxoid Unipath Ltd, Hampshire, UK), and Wallerstein Laboratory nutrient agar medium (WL medium; Oxoid Unipath Ltd, Hampshire, UK) (Pallman et al., 2001). The latter was supplemented with biphenyl
Table 1 Details of the fermentation trials carried out in the present study Inoculated strain of Saccharomyces cerevisiae
Barrels Without mother sediment
With mother sediment
Strain SS1 (DISAABA) Strain SS2 (DISAABA) Strain 1545 (DBVPG) Strain “Sherry Yeast” (Unican) Control (without inoculum)
F1 F2 F3 F4 F5
F6 F7 F8 F9 F10
3. Results and discussion 3.1. Effects of mother sediment on yeast growth and ethanol production Viable plate counts of the ten barrels were carried out at three sampling times on both WL and LA media (Fig. 1). WL is a differential medium that allows the preliminary identification of wine yeasts on the basis of the colour and morphology of their colonies (Pallman et al., 2001). This medium was therefore used both to evaluate the viable yeast population and the yeast biodiversity in Vinsanto, and to differentiate the Saccharomyces type colonies. LA is a selective medium that does not support the growth of S. cerevisiae (Lin, 1975). The viable plate counts for the non-Saccharomyces population were carried out on this medium. The results obtained immediately after barrel filling (time 0) indicated that the number of Saccharomyces type colonies varied from 1 × 105 to 1.2 × 106 CFU ml− 1 in the inoculated barrels. This variability in viable counts could be due to the high sugar concentration of the must that might have determined different rates of cell death upon inoculation but it could be also a consequence of experimental variability. No Saccharomyces type colonies were detected by viable plate counts in the non-inoculated barrels, with or without the mother sediment. At time 1 (three months after inoculation), in the barrels inoculated with the starter strains and in the presence of the mother sediment, the Saccharomyces type colonies had increased their initial concentrations 5-fold to 30-fold, while an increase of 0.7-fold to 9-fold was seen for the samples without mother sediment (Fig. 1). A similar trend was seen in the non-inoculated barrels F5 and F10 where, in presence of the mother sediment (F10), the number of Saccharomyces type colonies was two orders of magnitude higher than in the corresponding samples without
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Fig. 1. Viable plate counts of Saccharomyces (□) and non-Saccharomyces (○) yeasts from the fermentation trials carried out with and without addition of mother sediment.
mother sediment (F5) (4.5 × 106 CFU ml− 1 and 9 × 104 CFU ml− 1, respectively). Thus, the addition of the mother sediment provided a positive effect on S. cerevisiae growth in natural and inoculated fermentations. At time 2 (five months after inoculation), when a further increase in ethanol concentrations was seen, no more Saccharomyces type colonies were detected on the plates. The non-Saccharomyces yeasts (Fig. 1) were present at concentrations from 8 × 105 CFU ml− 1 to 1.3 × 106 CFU ml− 1 at time 0. After three months (time 1), in spite of the high ethanol concentrations (11.2%– 13.6%, v/v), about 4–7 × 105 CFU ml− 1 of non-Saccharomyces yeasts were still present in the majority of the fermentation trials, while there was a marked decrease in trials F2 and F5 (both without mother sediment) in which 4 × 104 CFU ml− 1 and 1.2 × 105 CFU ml− 1 were counted, respectively. On the basis of morphological features nonSaccharomyces yeasts, mainly ascribed to the Zygosaccharomyces genus, were still present at time 2 in the trials F7, F9 and F10 (all with mother sediment). As already seen in a previous study (Domizio et al., 2007), the mother sediment can serve as a source of these yeasts, and their persistence in Vinsanto wines at time 2 could be explained by their adaptation to the Vinsanto conditions and their well known tolerance to high ethanol and sugar concentrations. The mother sediment also showed a positive effect on fermentative performance, as its presence in the barrels was always accompanied by a significant increase in ethanol production (Table 2). Other studies have shown that wine yeasts can incorporate exogenous lipids under anaerobic conditions (Luparia et al., 2004) and use them to modulate
Table 2 Ethanol, residual sugar and volatile acidity of Vinsanto wines fermented with and without mother sediment at sampling time 2 Barrels
Ethanol (%v/v)
Residual sugar (% w/v)
Volatile acidity (g/l acetic acid)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
13.35 12.38 12.16 12.54 11.96 15.20 15.58 15.15 14.70 13.43
12.63 14.31 14.67 13.75 15.00 10.07 9.07 9.91 10.20 12.64
0.90 0.41 0.61 0.65 0.43 1.35 0.96 1.16 0.90 0.56
the lipid composition of their cell membranes in response to environmental stimuli (Belviso et al., 2004). Thus, the observed effects of the mother sediment on yeast growth and ethanol production could be seen as a consequence of the use of these lipids that are released following yeast autolysis (Pueyo et al., 2000). The trials carried out in the presence of mother sediment showed higher volatile acidities, although within the normal range for this type of wines. This could be due to the persistence of Zygosaccharomyces yeasts, well known producers of acetic acid (Giudici and Zambonelli, 1992). 3.2. Effects of mother sediment on biodiversity of S. cerevisiae At time 1, when about 55% of the total sugar was fermented, ten Saccharomyces type colonies for each inoculated barrel and the S. cerevisiae strains used as fermentation starters were subjected to molecular characterization. For this purpose, PCR amplification was carried out for the HSP150 and SED1 genes (Marinangeli et al., 2004) and the inter-δ sequences (Legras and Karst, 2003), and the molecular profile of each starter was compared with those of the isolates coming from the same barrel. The analyses of the amplification profiles of the target sequences proved to be useful for the separation of the Saccharomyces type colonies and the inoculated starters and the results obtained indicate that the starter Unican Sherry Yeast was the only one to dominate the wild microflora. In particular, seven out of the ten isolates of barrel F4 (without mother sediment) and all of the isolates of barrel F9 (with mother sediment) had the same molecular profile for the starters, with each of the primer pairs used (Fig. 2). Of the remaining strains, only SS1 showed a limited persistence during the fermentation process, while SS2 and DBVPG 1545 could not be reisolated from the inoculated barrels (data not shown). At the same time, ten Saccharomyces type colonies isolated from the two noninoculated barrels (F5 and F10) were subjected to molecular characterization. The results obtained indicated the presence of three and five different biotypes in barrels F5 and F10, without mother sediment and with mother sediment, respectively (Fig. 3). The amplification profiles generated by all of the Saccharomyces type colonies that were isolated from the ten trials and differed from the starter biotypes (from now on indicated as wild Saccharomyces) showed the existence of 10 and 17 different biotypes in barrels without and with mother sediment, respectively (Fig. 3). Interestingly, in barrels F3 and F8, where none of the wild Saccharomyces showed
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Fig. 2. Molecular characterization of the Saccharomyces type colonies isolated from barrels F4 (without mother sediment) and F9 (with mother sediment) inoculated with starter Unican Sherry yeasts. SY: starter yeast. Lanes 1–10: isolates 1–10. a–bindicate different amplification profiles. L: Gene Ruler™ DNA Ladder Mix (MBI Fermentas); L1O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
the same molecular profile of the starter strain inoculated, a situation similar to that of barrels F5 and F10 was seen. Eight commercial starter strains that are commonly used in wineries were subjected to molecular characterization and compared with those of isolates from the ten barrels. None of the inoculated strains showed the same amplification profile of the wild Saccharomyces. Therefore these last did not seem to derive from cross contamination of the barrels with any of the starter strain used in the winery (Fig. 4). Thus, the biodiversity seen in the natural and inoculated fermentations in the presence of mother sediment might be a consequence of its addition. Domizio et al. (2007) reported that mother sediment does not appear to contain yeasts belonging to the species S. cerevisiae. Thus, the effects of mother sediment on S. cerevisiae growth and biodiversity could be due to its content of lipids and other nutritional factors, which would support mainly the growth of the S. cerevisiae strains naturally present in the must and selected in peculiar environments such as dried grapes, fruttaio and vinsantaia.
Fig. 3. Amplification of inter-δ sequences of the wild Saccharomyces isolated from barrels with and without mother sediment. The numbers given above each lane indicate different strains within each barrel. L1: O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
Interestingly, the addition of mother sediment did not have any effect on the dominance of the inoculated starters. Accordingly, the only strain that dominated the wild microflora (Unican Sherry yeast) did so irrespective of the absence or presence of mother sediment in the barrel. The inability to dominate of the other starters inoculated might be explained considering that fermentation occurs under stressful conditions during Vinsanto winemaking. Indeed, the musts used for the production of Vinsanto had sugar concentrations higher than 26%, and the resulting wines contained high ethanol concentrations at the end of fermentation (≥16%, v/v). In addition, it needs to be noted that the inoculation of the starter strains occurred in February. Thus, at the beginning of fermentation, the yeasts were subjected to low temperatures. These conditions might have negatively affected the dominance of the starter strains that had not been subjected to selective pressure in fermentation processes with similar characteristics. In agreement with this hypothesis the Unican Sherry yeast is normally used for the production of sherry wines in fermentative processes with characteristics similar to those of Vinsanto making. In conclusion, the addition of mother sediment to the must has a positive effect on ethanol production and favours the implantation of wild Saccharomyces and non Saccharomyces strains during fermentation. Since the fermentative activity of wine yeasts with different technological capabilities can improve the complexity of taste and other desirable characteristics of wine (Grossmann et al.,1996; Cheraiti
Fig. 4. Amplification of inter-δ sequences of the eight commercial starter strains (lanes A to H). Asterisks indicate strains that present different amplification profiles with HSP150 and SED1 primers. L1: O' Gene Ruler™ DNA Ladder Mix (MBI Fermentas).
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et al., 2005, Domizio et al., 2007), the addition of mother sediment to the must can be considered an important practice for the improvement of the final quality of Vinsanto. Acknowledgements The authors wish to thank Marchese Antinori s.r.l., who provided the wine cellar facilities and the financial support for the realization of this study, Marilena Budroni for providing strains SS1 and SS2, and Cristina Bianchi for her valuable contribution to the molecular analyses. References Belviso, S., Bardi, L., Biondi Bartolini, A., Marzona, M., 2004. Lipid nutrition of Saccharomyces cerevisiae in winemaking. Canadian Journal of Microbiology 50, 669–674. Cheraiti, N., Guezenec, S., Salmon, J.M., 2005. Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions. Applied and Environmental Microbiology 71 (1), 255–260. Domizio, P., Lencioni, L., Ciani, M., Di Blasi, S., Pontremolesi, C., Sabatelli, M.P., 2007. Spontaneous and inoculated yeast populations dynamics and their effect on organoleptic characters of Vinsanto wine under different process conditions. International Journal of Food Microbiology 115, 281–289. Giudici, P., Zambonelli, C., 1992. Biometric and genetic study on acetic acid production for breeding of wine yeast. American Journal of Enology and Viticulture 43, 370–374.
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