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Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic... Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homo- geneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage. . . . . . Keywords Spontaneous fermentation Yeasts Microbial selection Biodiversity Biodynamic Syrah Findings (Capozzi et al. 2015). It is common to observe a yeast popu- lation of around 10 cells/g on ripe bunches and on grape In the last few years, there has undoubtedly been growing must, composed mainly of yeasts not belonging to the genus interest among consumers regarding wines with features more Saccharomyces (Barata et al. 2012). It is also widely known closely linked to their areas of origin. This trend has led that an increase in ethanol content, due to alcoholic fermenta- winemakers to rethink the approach to alcoholic fermentation, tion, leads to the selection of microbiota with a prevalence of avoiding the use of selected yeast and entrusting the fermen- Saccharomyces cerevisiae, after accumulation of 5–6% v/v tation process to microbiota naturally present in the cellar and ethanol in fermenting grape must. The use of selected strains on the grapes. This approach might seem a step back in terms of S. cerevisiae does not alter the evolution of wine microbi- of the scientific and technological state of the art of oenology, ota, but accelerates it, encouraging a faster increase in the but given its increasing diffusion and economic significance, alcohol content of grape must (Guzzon et al. 2014;Ciani it deserves attention and further scientific study. Grapes and et al. 2016). However, if Bspontaneous^ fermentation is well cellar equipment are populated by a wide range of yeasts and managed from the technological and analytical point of view, bacteria that evolve during the production process, according it is possible to obtain wines qualitatively comparable with to the environmental conditions and technological choices those obtained by inoculating active dry yeasts (Chaves- López et al. 2009). The risk of microbial spoilage in the case of alcoholic fermentation performed without the use of active * Raffaele Guzzon dry yeast could be associated with incomplete knowledge of raffaele.guzzon@fmach.it the specific features of the native microbiota in each specific oenological environment and, consequently, incorrect techno- Centro di Trasferimento Tecnologico, Fondazione Edmund Mach, logical approaches. The study of biodiversity associated with Via Mach 1, 38010 San Michele all’Adige, TN, Italy spontaneous fermentation, and oenological characterisation of Department of Agriculture, Food and Environment (DAFE), identified yeasts, contributes to increasing comprehension of University of Pisa, Via Borghetto 80, 56124 Pisa, Italy 964 Ann Microbiol (2018) 68:963–967 the microbial dynamics of winemaking, preventing the risk of 10 cfu/mL) revealed a large population of yeasts attributable wine spoilage. Many papers have already discussed this topic to the Saccharomyces genus, with a low population of other (Comitini et al. 2017). In this paper, we report on the study of a yeast genera of oenological interest. Furthermore, acetic acid S. cerevisiae population isolated during the winemaking pro- bacteria were not found (< 5 × 10 cfu/mL), while a popula- cess in a winery operating in the Cortona DOC area (southern tion of lactic acid bacteria potentially able to spoil wine (Liu Tuscany, Italy), following a biodynamic approach that ex- 2002) was present in all samples. The significant contamina- cludes the use of active dry yeast, avoiding the risk of con- tion by lactic acid bacteria during alcoholic fermentation, on tamination of the microbiota native to this scenario. average 9.3 ± 6.8 × 10 cfu/mL, was probably favoured by the The study began with the isolation of yeasts, carried out high pH of the grape must and the absence of SO . considering eight different samples of fermenting grape must One hundred yeast samples were isolated and purified on (Table 1), after degradation of 90% of initial sugar content. WL agar (Oxoid), on the basis of provenience (vineyard) and This differs from the approach frequently adopted in works morphology. The yeasts isolated were transferred into YM studying the biodiversity of the oenological environment broth (Oxoid) to encourage rapid growth, and after 3 days of (Settanni et al. 2012). The yeasts were isolated from incubation at 30 °C, total DNA was extracted and purified fermenting grape must having more than 10% ethanol, in or- using the Insta Gene Matrix kit (Bio-Rad, CA). Strain typing der to identify yeast strains with the best ability to drive alco- was performed through analysis of interdelta sequences (ISA- holic fermentation, and resistance to wine limiting factors. PCR) (Legras and Karst 2003; Charpentier et al. 2009), The must was made from Syrah grapes, after gentle crushing, obtaining a discrimination of 11 different strains. Their appur- without sulphur dioxide and the addition of selected yeast. tenance to the S. cerevisiae species was confirmed by se- Yeasts were counted on WL agar medium (Oxoid, UK), ac- quencing the D1/D2 region of 26S rDNA using NL1 and cording to OIV standards (OIV 2016); determination of non- NL4 primers (Kurtzman and Robnett 1998). The identities Saccharomyces yeast (Agar Lysine, Oxoid), lactic acid bacte- (≥ 97%) of the sequences were verified with a BlastN ria (MRS agar, Oxoid) and acetic bacteria (ACTS agar, Oxoid) (Altschul et al. 1997)search against theNational Centrefor was also carried out following the same protocol. The results Biotechnology Information (NCBI) non-redundant sequence (Table 1) confirmed the hypothesis on the basis of sampling. database, located at http://www.ncbi.nlm.nih.gov. There was The difference in plate counts obtained on WL Agar (mean of no correlation between the strain and the vineyard lots from 8.0 ± 0.6 × 10 cfu/mL) and Lysine Agar (mean of 1.8 ± 0.9 × which the grapes came, with different strains widespread in Table 1 First section. Main features of the wines used to isolate S. cerevisiae strains. Second section. Results of oenological tests performed with S. cerevisiae strains First section. Main features of the wines used to sample S. cerevisiae strains Number of Date of sampling Density of Yeast Non-Saccharomyces Lactic acid bacteria vat must yeast (Days from start of AF) (ufc/mL) 1 26/09 (+ 6) 995 8.20E+07 2.10E+04 3.80E+04 3 4/10 (+ 5) 999 8.70E+07 3.80E+04 2.60E+04 4 27/09 (+ 8) 996 8.90E+07 1.30E+04 1.20E+05 6 05/10 (+ 8) 997 8.20E+07 1.20E+04 8.90E+04 8 30/09 (+ 7) 993 7.40E+07 2.20E+04 1.60E+05 11 30/09 (+ 7) 993 7.80E+07 1.60E+04 2.10E+05 13 03/10 (+ 11) 992 7.40E+07 1.10E+04 8.80E+04 15 29/09 (+ 9) 992 7.60E+07 1.40E+04 1.30E+04 Second section. Results of oenological tests performed with the 11 S. cerevisiae strains Grape must Progress of fermentation after Ethanol Sugars Volatile Malic acid Tot. Volatile Stuck AF 2/5/10 days acidity SO phenols % (% vol) (g/L) (mg/L) Strain M1 8.5 ± 0.7 62.4 ± 2.6 96.8 ± 3.6 13.0 ± 0.7 3.89 ± 1.39 0.24 ± 0.06 2.80 ± 0.01 35 ± 6 202 ± 103 1, 8 M2 8.7 ± 0.8 53.9 ± 6.0 94.7 ± 4.4 13.2 ± 0.7 3.68 ± 1.34 0.30 ± 0.10 2.61 ± 0.13 42 ± 8 228 ± 104 5, 6, 8, 10, 11 M3 6.6 ± 0.9 56.5 ± 8.6 94.9 ± 5.7 14.2 ± 0.7 3.04 ± 1.1 0.38 ± 0.15 2.31 ± 0.12 46 ± 3 236 ± 91 2, 3, 5, 6, 8, 10, 11 (Mean ± SD n:11). Grape must 1: sugars 220 g/L, pH 3.24, YAN 164 mg/L; grape must 2: sugars 230 g/L, pH 3.58, YAN 137 mg/L; grape must 3: sugars 250 g/L, pH 3.81, YAN 118 mg/L Sugar value was calculated considering strains that accomplish alcoholic fermentation, with sugar residue below 5 g/L. Ann Microbiol (2018) 68:963–967 965 the entire set of samples. This result is reasonable considering to monitor sulphur dioxide. Volatile phenols were quantified the small size of the winery, and the need to use the same using HPLC (Agilent Technologies, CA), equipped with a oenological equipment, with the occurrence of cross- colorimetric array detector as proposed by Larcher et al. contamination. However, the biodiversity observed in the S. (2007). From the kinetic point of view, we chose to focus cerevisiae species was quite high, despite the uniformity of the our attention on three moments: the lag phase (2 days after features in the isolation source and the small size of the yeast inoculum), the end of the exponential phase (5 days after vineyard (about 8 ha, located in the same area). These yeast inoculum), and complete fermentation (arbitrarily observations agree with the results of previous studies established at 10 days after yeast inoculum); the progress of carried out in wineries that operate using biodynamic alcoholic fermentation was expressed as a percent of the the- processes (Morrison-Whittle et al. 2017). Pure strains, named oretical total weight loss (Fig. 1a). The initial yeast inoculum from A1 to A11, were stored in a suitable synthetic medium was set at 10 cfu/mL, ensuring the prompt start of alcoholic (YM + 20% of glycerol, Carlo Erba, I) at − 80 °C. fermentation in all tests, with a mean weight loss of 7.9 ± 1.3% Physiological tests were carried out in order to evaluate the after 2 days (separate data for grape must in Table 1). After oenological performance of the 11 S. cerevisiae strains, and 5 days, the first differences were already highlighted. The the possible presence of spoilage characteristics, such as poor mean weight loss was 59.1 ± 6.5%, but 4 strains—A2, A3, fermentative activity, excessive production of acetic acid, sul- A8 and A11—showed progress in alcoholic fermentation be- phur dioxide or volatile phenols (Guzzon et al. 2014). The low the mean for the population. A similar trend was observed three grape musts considered had sugar content between 220 after 10 days’ fermentation for three strains that showed per- and 240 g/L, a pH between 3.24 and 3.81 and YAN between formance below the mean (96.0 ± 3.8%). The measurement of 164 and 118 mg/L, and they were supplemented with 500 mg/ residual sugars in the wine obtained (Table 1) showed that Lof p-cumaric acid (Sigma-Aldrich, MO) to stimulate vinyl strains A4, A7 and A9 achieved alcoholic fermentation in phenol production. Cellular growth was monitored with plate the three tests, in wines with less than 5 g/L of sugars. As counts; the evolution of alcoholic fermentation was followed expected, most of the problems in achieving alcoholic fermen- by measuring the weight loss of samples due to CO produc- tation were observed at the highest potential ethanol content tion. The main chemical parameters of the wines obtained (grape must 3), with three strains—A8, A10 and A11—that were determined with FT-IR spectroscopy using a Wine were unable to completely degrade sugars in the full set of Scan (Foss) apparatus and a titrator (Crison Instrument, ES) tests. This observation is particularly significant because the Fig. 1 a Sugar consumption (expressed as %) of a population of 11 by the ISA-PCR products of S. cerevisiae isolates at the end of fermen- S. cerevisiae strains in tests performed in three different grape musts tation performed during the 2012 harvest. We observed the presence of with increasingly harsh conditions. M1: sugars 220 g/L, pH 3.24, YAN some strains isolated and characterised through this work (lanes 1, 2, 3 164 mg/L; M2: sugars 230 g/L, pH 3.58, YAN 137 mg/L; M3: sugars and 4) and new indigenous strains (lanes 4, 6, 8 and 10) 250 g/L, pH 3.81, YAN 118 mg/L. b Electrophoresis patterns generated 966 Ann Microbiol (2018) 68:963–967 problem of incomplete consumption of sugars, with the con- in the microbial population of the winery. Figure 1b shows the sequential possibility of developing spoilage microorganisms electrophoretic pattern of S. cerevisiae strains identified at the such as Brettanomyces or lactic acid bacteria (Chatonnet et al. end of alcoholic fermentation performed in the second year 1995; Loureiro and Malfeito-Ferreira 2003), has been more and inoculated with the mixture of strains selected in the first frequent in recent years, due to climate change and the part of work. Strain typing was performed with ISA-PCR resulting higher sugar content of grapes. The potential value according to the previous experiment. The presence of 8 dif- of 15% alcohol established in grape must sample 3 is not ferent biotypes confirmed the wide biodiversity observed in unusual in the Mediterranean oenological area. It is therefore this oenological scenario. Of the strains inoculated, four were important to underline that native yeast strains, developing in identified in lanes 1, 2, 3 and 4 (Fig. 1b). Some new the presence of specific environmental factors, are not always S. cerevisiae strains (lanes 4, 6, 8 and 10 in Fig. 1b) involved suitable for guaranteeing efficient alcoholic fermentation. in the fermentative process were also found. Spontaneous fermentation must be adequately monitored with The results obtained in this work confirm that there are microbiological assays designed to provide rapid and reliable S. cerevisiae strains of promising technological value in the information about the physiological state of the yeast popula- spontaneous microbiota characterising the winemaking pro- tion (Guzzon and Larcher 2015; OIV 2016). On the other cess in a biodynamic winery, capable of guaranteeing efficient hand, it was possible to isolate at least three strains of biotransformation and potentially high-quality wines. S. cerevisiae endowed with good fermentative activity and However, it is crucial to apply a technological approach suit- high resistance to ethanol from the complex microbial popu- able for stimulating these strains within a complex microbiota lation present in fermenting grape must samples. The three that also contains spoilage microorganisms such as lactic acid spoilage characteristics taken into consideration, the produc- bacteria or yeast with poor fermentative activity. In this way, tion of acetic acid, sulphur dioxide and volatile phenols, strains of high oenological suitability are able to drive alco- showed different trends (Table 1). The accumulation of acetic holic fermentation without eliminating the biodiversity char- acid (mean of 0.3 ± 0.1 g/L) was generally low, considering acteristic of each harvest. the high sugar content of grape must samples, which induces Acknowledgements We would like to thank Stefano Amerighi, Giulia osmotic stress and accumulation of acetic acid (Bely et al. Marangon, Alessandro Magrin and Calogero Caruana for their sugges- 2008; Teixeira et al. 2011). The production of 222 ± 98 mg/ tions and for stimulating discussion throughout the course of this work. L of volatile phenols, corresponding to a conversion rate of 44.5 ± 19.5%, was comparable to that of some S. cerevisiae Funding This work was funded with the standard resources of the strains used as fermentation starters (Guzzon et al. 2014), and Edmund Mach Foundation and the University of Pisa. acceptable for the production of red wines (Rojas et al. 2012). The accumulation of sulphur dioxide appeared to be more Compliance with ethical standards closely linked to the initial sugar content than to the features Conflicts of interest The authors declare that they have no conflict of of each S. cerevisiae strain (Table 1). However, strains A1 and interest. A2 produced the least over the full set of tests, while strains A9 and A10 accumulated the highest amount of sulphur diox- Research involving human participants and/or animals N/A ide in all wines. The level of SO reached at the end of fer- mentation of grape must 3 (46 ± 3 mg/L) could potentially Informed consent Informed consent was obtained from all the individ- lead to stuck malolactic fermentation; this aspect deserves ual participants involved in the study. particular consideration, because the combination of high eth- anol, pH and sulphur dioxide content in wines could stimulate the development of spoilage lactic acid bacteria such as Pediococcus spp. (Bartowsky 2009). References Isolated strains were employed in the subsequent vintage to Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, drive alcoholic fermentation, to avoid an excessive lag-phase Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new gener- which could cause uncontrolled proliferation of alterative mi- ation of protein database search programs. Nucleic Acids Res 25: croorganisms (Renouf 2015). The strains were inoculated by 3389–3402 preparing a pied de cuve before the harvest, having a volume Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of 1% of the overall mass of grapes harvested. At the end of of wine grape berries. Int J Food Microbiol 153:243–259 Bartowsky EJ (2009) Bacterial spoilage of wine and approaches to min- alcoholic fermentation, yeasts belonging to the S. cerevisiae imize it. Lett Appl Microbiol 48:149–156 species were isolated and characterised at strain level, accord- Bely M, Stoeckle P, Masneuf-Pomarede I, Dubourdieu D (2008) Impact ing to the previously described experiments. The objective of of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture this second set of microbiological assays was to verify the on high-sugar fermentation. Int J Food Microbiol 122:312–320 capacity of isolated strains of S. cerevisiae to remain active Ann Microbiol (2018) 68:963–967 967 Capozzi V, Garofalo C, Chiriatti MA, Grieco F, Spano G (2015) Larcher R, Nicolini G, Puecher C, Bertoldi D, Moser S, Favaro G (2007) Determination of volatile phenols in wine using high-performance Microbial terroir and food innovation: the case of yeast biodiversity in wine. Microbiol Res 181:75–83 liquid chromatography with a coulometric array detector. Anal Chim Charpentier C, Colin A, Alais A, Legras JL (2009) French Jura flor Acta 582:55–60 yeasts: genotype and technological diversity. Antonie van Legras JL, Karst F (2003) Optimisation of interdelta analysis for Leeuwenhoek 95:263–273 Saccharomyces cerevisiae strain characterisation. FEMS Microbiol Chatonnet P, Dubourdieu D, Boidron JN (1995) The influence of Lett 221:249–255 Brettanomyces/Dekhera spp. yeasts and lactic acid bacteria on the Liu SQ (2002) Malolactic fermentation in wine - beyond deacidification. ethylphenol content of red wines. Am J Enol Vitic 46:463–468 J Appl Microbiol 92:589–601 Chaves-López C, Serio A, Osorio-Cadavid E, Paparella A, Suzzi G Loureiro V, Malfeito-Ferreira M (2003) Spoilage yeasts in the wine in- (2009) Volatile compounds produced in wine by Colombian wild dustry. Int J Food Microbiol 86:23–50 Saccharomyces cerevisiae strains. Ann Microbiol 59:733–740 Morrison-Whittle P, Lee SA, Goddard MR (2017) Fungal communities Ciani M, Capece A, Comitini F, Canonico L, Siesto G, Romano P (2016) are differentially affected by conventional and biodynamic agricul- Yeast interactions in inoculated wine fermentation. Front Microbiol tural management approaches in vineyard ecosystems. Agric article no. 555 Ecosyst Environ 246:306–313 Comitini F, Capece A, Ciani M, Romano P (2017) New insights on the OIV (2016) Compendium of international methods of analysis of wines use of wine yeasts. Curr Opin Food Sci 13:44–49 and musts (vol 2) OIV-MA-AS4-01. OIV, Paris Guzzon R, Larcher R (2015) The application of flow cytometry in micro- Renouf V (2015) Brettanomyces et phénols volatils. Lavoisier, Paris biological monitoring during winemaking: two case studies. Ann Rojas IB, Smith PA, Bartowsky EJ (2012) Influence of choice of yeasts Microbiol 65:1865–1878 on volatile fermentation-derived compounds, colour and phenolics Guzzon R, Nicolini G, Nardin T, Malacarne M, Larcher R (2014) Survey composition in Cabernet Sauvignon wine. J Microbiol Biotechnol about the microbiological features, the oenological performance and 28:3311–3321 the influence on the character of wine of active dry yeast employed Settanni L, Sannino C, Francesca N, Guarcello R, Moschetti G (2012) as starters of wine fermentation. Int J Food Sci Technol 49:2142– Yeast ecology of vineyards within Marsala wine area (western 2148 Sicily) in two consecutive vintages and selection of autochthonous Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of asco- Saccharomyces cerevisiae strains. J Biosci Bioeng 114:606–614 mycetous yeasts from analysis of nuclear large subunit (26S) ribo- Teixeira MC, Mira NP, Sa-Correia I (2011) A genome-wide perspective somal DNA partial sequences. Antonie van Leeuwenhoek Int J Gen on the response and tolerance to food-relevant stresses in Mol Microbiol 73:331–371 Saccharomyces cerevisiae. Curr Opin Biotechnol 22:150–156 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-018-1405-4
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Abstract

Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homo- geneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage. . . . . . Keywords Spontaneous fermentation Yeasts Microbial selection Biodiversity Biodynamic Syrah Findings (Capozzi et al. 2015). It is common to observe a yeast popu- lation of around 10 cells/g on ripe bunches and on grape In the last few years, there has undoubtedly been growing must, composed mainly of yeasts not belonging to the genus interest among consumers regarding wines with features more Saccharomyces (Barata et al. 2012). It is also widely known closely linked to their areas of origin. This trend has led that an increase in ethanol content, due to alcoholic fermenta- winemakers to rethink the approach to alcoholic fermentation, tion, leads to the selection of microbiota with a prevalence of avoiding the use of selected yeast and entrusting the fermen- Saccharomyces cerevisiae, after accumulation of 5–6% v/v tation process to microbiota naturally present in the cellar and ethanol in fermenting grape must. The use of selected strains on the grapes. This approach might seem a step back in terms of S. cerevisiae does not alter the evolution of wine microbi- of the scientific and technological state of the art of oenology, ota, but accelerates it, encouraging a faster increase in the but given its increasing diffusion and economic significance, alcohol content of grape must (Guzzon et al. 2014;Ciani it deserves attention and further scientific study. Grapes and et al. 2016). However, if Bspontaneous^ fermentation is well cellar equipment are populated by a wide range of yeasts and managed from the technological and analytical point of view, bacteria that evolve during the production process, according it is possible to obtain wines qualitatively comparable with to the environmental conditions and technological choices those obtained by inoculating active dry yeasts (Chaves- López et al. 2009). The risk of microbial spoilage in the case of alcoholic fermentation performed without the use of active * Raffaele Guzzon dry yeast could be associated with incomplete knowledge of raffaele.guzzon@fmach.it the specific features of the native microbiota in each specific oenological environment and, consequently, incorrect techno- Centro di Trasferimento Tecnologico, Fondazione Edmund Mach, logical approaches. The study of biodiversity associated with Via Mach 1, 38010 San Michele all’Adige, TN, Italy spontaneous fermentation, and oenological characterisation of Department of Agriculture, Food and Environment (DAFE), identified yeasts, contributes to increasing comprehension of University of Pisa, Via Borghetto 80, 56124 Pisa, Italy 964 Ann Microbiol (2018) 68:963–967 the microbial dynamics of winemaking, preventing the risk of 10 cfu/mL) revealed a large population of yeasts attributable wine spoilage. Many papers have already discussed this topic to the Saccharomyces genus, with a low population of other (Comitini et al. 2017). In this paper, we report on the study of a yeast genera of oenological interest. Furthermore, acetic acid S. cerevisiae population isolated during the winemaking pro- bacteria were not found (< 5 × 10 cfu/mL), while a popula- cess in a winery operating in the Cortona DOC area (southern tion of lactic acid bacteria potentially able to spoil wine (Liu Tuscany, Italy), following a biodynamic approach that ex- 2002) was present in all samples. The significant contamina- cludes the use of active dry yeast, avoiding the risk of con- tion by lactic acid bacteria during alcoholic fermentation, on tamination of the microbiota native to this scenario. average 9.3 ± 6.8 × 10 cfu/mL, was probably favoured by the The study began with the isolation of yeasts, carried out high pH of the grape must and the absence of SO . considering eight different samples of fermenting grape must One hundred yeast samples were isolated and purified on (Table 1), after degradation of 90% of initial sugar content. WL agar (Oxoid), on the basis of provenience (vineyard) and This differs from the approach frequently adopted in works morphology. The yeasts isolated were transferred into YM studying the biodiversity of the oenological environment broth (Oxoid) to encourage rapid growth, and after 3 days of (Settanni et al. 2012). The yeasts were isolated from incubation at 30 °C, total DNA was extracted and purified fermenting grape must having more than 10% ethanol, in or- using the Insta Gene Matrix kit (Bio-Rad, CA). Strain typing der to identify yeast strains with the best ability to drive alco- was performed through analysis of interdelta sequences (ISA- holic fermentation, and resistance to wine limiting factors. PCR) (Legras and Karst 2003; Charpentier et al. 2009), The must was made from Syrah grapes, after gentle crushing, obtaining a discrimination of 11 different strains. Their appur- without sulphur dioxide and the addition of selected yeast. tenance to the S. cerevisiae species was confirmed by se- Yeasts were counted on WL agar medium (Oxoid, UK), ac- quencing the D1/D2 region of 26S rDNA using NL1 and cording to OIV standards (OIV 2016); determination of non- NL4 primers (Kurtzman and Robnett 1998). The identities Saccharomyces yeast (Agar Lysine, Oxoid), lactic acid bacte- (≥ 97%) of the sequences were verified with a BlastN ria (MRS agar, Oxoid) and acetic bacteria (ACTS agar, Oxoid) (Altschul et al. 1997)search against theNational Centrefor was also carried out following the same protocol. The results Biotechnology Information (NCBI) non-redundant sequence (Table 1) confirmed the hypothesis on the basis of sampling. database, located at http://www.ncbi.nlm.nih.gov. There was The difference in plate counts obtained on WL Agar (mean of no correlation between the strain and the vineyard lots from 8.0 ± 0.6 × 10 cfu/mL) and Lysine Agar (mean of 1.8 ± 0.9 × which the grapes came, with different strains widespread in Table 1 First section. Main features of the wines used to isolate S. cerevisiae strains. Second section. Results of oenological tests performed with S. cerevisiae strains First section. Main features of the wines used to sample S. cerevisiae strains Number of Date of sampling Density of Yeast Non-Saccharomyces Lactic acid bacteria vat must yeast (Days from start of AF) (ufc/mL) 1 26/09 (+ 6) 995 8.20E+07 2.10E+04 3.80E+04 3 4/10 (+ 5) 999 8.70E+07 3.80E+04 2.60E+04 4 27/09 (+ 8) 996 8.90E+07 1.30E+04 1.20E+05 6 05/10 (+ 8) 997 8.20E+07 1.20E+04 8.90E+04 8 30/09 (+ 7) 993 7.40E+07 2.20E+04 1.60E+05 11 30/09 (+ 7) 993 7.80E+07 1.60E+04 2.10E+05 13 03/10 (+ 11) 992 7.40E+07 1.10E+04 8.80E+04 15 29/09 (+ 9) 992 7.60E+07 1.40E+04 1.30E+04 Second section. Results of oenological tests performed with the 11 S. cerevisiae strains Grape must Progress of fermentation after Ethanol Sugars Volatile Malic acid Tot. Volatile Stuck AF 2/5/10 days acidity SO phenols % (% vol) (g/L) (mg/L) Strain M1 8.5 ± 0.7 62.4 ± 2.6 96.8 ± 3.6 13.0 ± 0.7 3.89 ± 1.39 0.24 ± 0.06 2.80 ± 0.01 35 ± 6 202 ± 103 1, 8 M2 8.7 ± 0.8 53.9 ± 6.0 94.7 ± 4.4 13.2 ± 0.7 3.68 ± 1.34 0.30 ± 0.10 2.61 ± 0.13 42 ± 8 228 ± 104 5, 6, 8, 10, 11 M3 6.6 ± 0.9 56.5 ± 8.6 94.9 ± 5.7 14.2 ± 0.7 3.04 ± 1.1 0.38 ± 0.15 2.31 ± 0.12 46 ± 3 236 ± 91 2, 3, 5, 6, 8, 10, 11 (Mean ± SD n:11). Grape must 1: sugars 220 g/L, pH 3.24, YAN 164 mg/L; grape must 2: sugars 230 g/L, pH 3.58, YAN 137 mg/L; grape must 3: sugars 250 g/L, pH 3.81, YAN 118 mg/L Sugar value was calculated considering strains that accomplish alcoholic fermentation, with sugar residue below 5 g/L. Ann Microbiol (2018) 68:963–967 965 the entire set of samples. This result is reasonable considering to monitor sulphur dioxide. Volatile phenols were quantified the small size of the winery, and the need to use the same using HPLC (Agilent Technologies, CA), equipped with a oenological equipment, with the occurrence of cross- colorimetric array detector as proposed by Larcher et al. contamination. However, the biodiversity observed in the S. (2007). From the kinetic point of view, we chose to focus cerevisiae species was quite high, despite the uniformity of the our attention on three moments: the lag phase (2 days after features in the isolation source and the small size of the yeast inoculum), the end of the exponential phase (5 days after vineyard (about 8 ha, located in the same area). These yeast inoculum), and complete fermentation (arbitrarily observations agree with the results of previous studies established at 10 days after yeast inoculum); the progress of carried out in wineries that operate using biodynamic alcoholic fermentation was expressed as a percent of the the- processes (Morrison-Whittle et al. 2017). Pure strains, named oretical total weight loss (Fig. 1a). The initial yeast inoculum from A1 to A11, were stored in a suitable synthetic medium was set at 10 cfu/mL, ensuring the prompt start of alcoholic (YM + 20% of glycerol, Carlo Erba, I) at − 80 °C. fermentation in all tests, with a mean weight loss of 7.9 ± 1.3% Physiological tests were carried out in order to evaluate the after 2 days (separate data for grape must in Table 1). After oenological performance of the 11 S. cerevisiae strains, and 5 days, the first differences were already highlighted. The the possible presence of spoilage characteristics, such as poor mean weight loss was 59.1 ± 6.5%, but 4 strains—A2, A3, fermentative activity, excessive production of acetic acid, sul- A8 and A11—showed progress in alcoholic fermentation be- phur dioxide or volatile phenols (Guzzon et al. 2014). The low the mean for the population. A similar trend was observed three grape musts considered had sugar content between 220 after 10 days’ fermentation for three strains that showed per- and 240 g/L, a pH between 3.24 and 3.81 and YAN between formance below the mean (96.0 ± 3.8%). The measurement of 164 and 118 mg/L, and they were supplemented with 500 mg/ residual sugars in the wine obtained (Table 1) showed that Lof p-cumaric acid (Sigma-Aldrich, MO) to stimulate vinyl strains A4, A7 and A9 achieved alcoholic fermentation in phenol production. Cellular growth was monitored with plate the three tests, in wines with less than 5 g/L of sugars. As counts; the evolution of alcoholic fermentation was followed expected, most of the problems in achieving alcoholic fermen- by measuring the weight loss of samples due to CO produc- tation were observed at the highest potential ethanol content tion. The main chemical parameters of the wines obtained (grape must 3), with three strains—A8, A10 and A11—that were determined with FT-IR spectroscopy using a Wine were unable to completely degrade sugars in the full set of Scan (Foss) apparatus and a titrator (Crison Instrument, ES) tests. This observation is particularly significant because the Fig. 1 a Sugar consumption (expressed as %) of a population of 11 by the ISA-PCR products of S. cerevisiae isolates at the end of fermen- S. cerevisiae strains in tests performed in three different grape musts tation performed during the 2012 harvest. We observed the presence of with increasingly harsh conditions. M1: sugars 220 g/L, pH 3.24, YAN some strains isolated and characterised through this work (lanes 1, 2, 3 164 mg/L; M2: sugars 230 g/L, pH 3.58, YAN 137 mg/L; M3: sugars and 4) and new indigenous strains (lanes 4, 6, 8 and 10) 250 g/L, pH 3.81, YAN 118 mg/L. b Electrophoresis patterns generated 966 Ann Microbiol (2018) 68:963–967 problem of incomplete consumption of sugars, with the con- in the microbial population of the winery. Figure 1b shows the sequential possibility of developing spoilage microorganisms electrophoretic pattern of S. cerevisiae strains identified at the such as Brettanomyces or lactic acid bacteria (Chatonnet et al. end of alcoholic fermentation performed in the second year 1995; Loureiro and Malfeito-Ferreira 2003), has been more and inoculated with the mixture of strains selected in the first frequent in recent years, due to climate change and the part of work. Strain typing was performed with ISA-PCR resulting higher sugar content of grapes. The potential value according to the previous experiment. The presence of 8 dif- of 15% alcohol established in grape must sample 3 is not ferent biotypes confirmed the wide biodiversity observed in unusual in the Mediterranean oenological area. It is therefore this oenological scenario. Of the strains inoculated, four were important to underline that native yeast strains, developing in identified in lanes 1, 2, 3 and 4 (Fig. 1b). Some new the presence of specific environmental factors, are not always S. cerevisiae strains (lanes 4, 6, 8 and 10 in Fig. 1b) involved suitable for guaranteeing efficient alcoholic fermentation. in the fermentative process were also found. Spontaneous fermentation must be adequately monitored with The results obtained in this work confirm that there are microbiological assays designed to provide rapid and reliable S. cerevisiae strains of promising technological value in the information about the physiological state of the yeast popula- spontaneous microbiota characterising the winemaking pro- tion (Guzzon and Larcher 2015; OIV 2016). On the other cess in a biodynamic winery, capable of guaranteeing efficient hand, it was possible to isolate at least three strains of biotransformation and potentially high-quality wines. S. cerevisiae endowed with good fermentative activity and However, it is crucial to apply a technological approach suit- high resistance to ethanol from the complex microbial popu- able for stimulating these strains within a complex microbiota lation present in fermenting grape must samples. The three that also contains spoilage microorganisms such as lactic acid spoilage characteristics taken into consideration, the produc- bacteria or yeast with poor fermentative activity. In this way, tion of acetic acid, sulphur dioxide and volatile phenols, strains of high oenological suitability are able to drive alco- showed different trends (Table 1). The accumulation of acetic holic fermentation without eliminating the biodiversity char- acid (mean of 0.3 ± 0.1 g/L) was generally low, considering acteristic of each harvest. the high sugar content of grape must samples, which induces Acknowledgements We would like to thank Stefano Amerighi, Giulia osmotic stress and accumulation of acetic acid (Bely et al. Marangon, Alessandro Magrin and Calogero Caruana for their sugges- 2008; Teixeira et al. 2011). The production of 222 ± 98 mg/ tions and for stimulating discussion throughout the course of this work. L of volatile phenols, corresponding to a conversion rate of 44.5 ± 19.5%, was comparable to that of some S. cerevisiae Funding This work was funded with the standard resources of the strains used as fermentation starters (Guzzon et al. 2014), and Edmund Mach Foundation and the University of Pisa. acceptable for the production of red wines (Rojas et al. 2012). The accumulation of sulphur dioxide appeared to be more Compliance with ethical standards closely linked to the initial sugar content than to the features Conflicts of interest The authors declare that they have no conflict of of each S. cerevisiae strain (Table 1). However, strains A1 and interest. A2 produced the least over the full set of tests, while strains A9 and A10 accumulated the highest amount of sulphur diox- Research involving human participants and/or animals N/A ide in all wines. 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Annals of MicrobiologySpringer Journals

Published: Nov 13, 2018

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