Effect of fermentation temperature on the microbial and physicochemical properties of silver carp sausages inoculated with Pediococcus pentosaceus

Food Research International 43 (2010) 773–779 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques Imma Andorrà a,1, Sara Landi b,1, Albert Mas a, Braulio Esteve-Zarzoso a,*, José M. Guillamón a,c a Biotecnologia Enològica, Departament de Bioquímica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i Virgili, Marcel
li Domingo s/n, 43007 Tarragona, Spain Department of Agricultural Science, University of Modena and Reggio Emilia, Via J. F. Kennedy, 17, 42100 Reggio Emilia, Italy c Departamento de Biotecnología de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, E-46100 Burjassot, València, Spain b a r t i c l e i n f o Article history: Received 26 May 2009 Accepted 18 November 2009 Keywords: DGGE Cloning QPCR Wine Grape Acetobacter aceti a b s t r a c t The population dynamics of micro-organisms during grape-must fermentation has been thoroughly studied. However, the main approach has relied on microbiological methods based on plating. This approach may overlook micro-organisms that (i) grow slowly or do not grow well on artificial media or (ii) whose population size is small enough to be detected by regular sampling. Culture-independent methods have been used and compared with the traditional plating method during wine fermentations performed at two different temperatures (13 °C and 25 °C). These methods include a qualitative technique, the DGGE; a semi-quantitative technique, the direct cloning of amplified DNA; and a quantitative technique, the QPCR. The biodiversity observed in the must and at the beginning of fermentation was much higher when DGGE or direct cloning were used. Quantification of the most frequent non-Saccharomyces yeast, Hanseniaspora uvarum and Candida zemplinina, showed that they survived throughout the fermentation process and, specifically, it revealed the quantitatively relevant presence of C. zemplinina until the end of fermentation. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The conversion of grape-must to wine is a complex biochemical process involving interactions between yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB). The metabolism of these micro-organisms contributes to the quality of the wine by releasing metabolites which are constituents of the flavour and aroma (Swiegers, Bartowsky, & Henschke, 2005). Wine microbiota is influenced by multiple factors which can be grouped into viticultural and oenological practices (Pretorius, 1999). The temperature of fermentation is an oenological factor which influences the evolution of wine populations (Fleet, 2003): the lower the temperature of fermentation, the higher the chance of survival of the non-Saccharomyces yeasts during alcoholic fermentation (Heard & Fleet, 1988; Sharf & Margalith, 1983). Likewise, Ribereau-Gayon, Dubourdieu, Donèche, and Lonvaud (2000) reported that low temperature notably reduced the growth of acetic and lactic acid bacteria. Low-temperature fermentations (below 15 °C) are considered to improve the wine’s aromatic profile. The increase in aroma may be related to a higher retention of volatile compounds. However, Beltran, Novo, Guillamón, Mas, and Rozès (2008) observed that this increase in flavour * Corresponding author. Tel.: +34 977 55 84 64; fax: +34 977 55 82 32. E-mail address: braulio.esteve@urv.cat (B. Esteve-Zarzoso). 1 Both authors contributed equally to this work. 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.11.014 and aroma was not only related to primary aroma retention. The evolution of yeast and bacteria species and their metabolism may also be involved in this improvement in the organoleptical characteristic of wines fermented at low temperature. Most previous studies on wine microbial ecology have invariably been conducted after the culture of the different micro-organisms in different media. Today, new culture-independent methods allow to identify and enumerate micro-organisms, avoiding the biases associated with traditional culture-dependent methods (Rantsiou et al., 2005). The presence of viable but non-culturable micro-organisms in wine samples has been described (Divol & Lonvaud-Funel, 2005; Millet & Lonvaud-Funel, 2000). These microorganisms are unable to grow on standard solid media within the laboratory but may justify the differences reported by various authors between isolated and naturally occurring species in wine samples (Cocolin & Mills, 2003; Hierro, Esteve-Zarzoso, González, Mas, & Guillamón, 2006; Mills, Johannsen, & Cocolin, 2002). The aim of this study was to analyse the evolution of wine microbial population during the fermentation of the same grapemust at low (13 °C) and optimum temperature for wine yeasts during fermentation (25 °C). Microbial populations were evaluated by using three culture-independent techniques: a qualitative technique (DGGE), a semi-quantitative technique (the direct cloning of amplified DNA) and a quantitative technique (the QPCR). DGGE and QPCR are two of the most widely used techniques for culture- 774 I. Andorrà et al. / Food Research International 43 (2010) 773–779 independent microbial analysis. In a previous study (Andorrà, Landi, Mas, Guillamón, & Esteve-Zarzoso, 2008), we enumerated the main wine microbial groups (yeast, lactic acid bacteria and acetic acid bacteria) using QPCR. In addition, we employed specific primers for the enumeration of two of the main yeast genera, Saccharomyces and Hanseniaspora. In the present study, we have also designed a new pair of primers for the enumeration of what is probably the third main wine yeast Candida stellata, or its current classification as Candida zemplinina (Sipiczki, Ciani, & Csoma, 2005). Moreover, in parallel to the analysis of species diversity by DGGE, we have evaluated the richness in yeast species through a direct amplification of DNA purified from wine samples and further cloning and identification of the amplicons. This technique has the additional advantage of making it possible to detect the relative abundance of the different species. To our knowledge, this is the first time that yeast diversity has been analysed using this strategy, thus avoiding some of the problems of cultivability of wine micro-organisms. 2.3. DNA extraction 2. Materials and methods 2.1. Reference strains and culture conditions The reference strains used in this study are listed in Table 1. Yeast were grown in YPD (2% glucose, 2% peptone, 1% yeast extract), lactic acid bacteria were grown in MRS (Oxoid, Hampshire, UK) and acetic acid bacteria were grown in Glucose media (5% glucose, 1% yeast extract). DNA from reference strains or wine samples was extracted according to Hierro et al. (2006) and diluted to 1–50 ng/lL. The concentration and purity of DNA was determined using a GenQuant spectrophotometer (Pharmacia, Cambridge, UK). Sample DNA was extracted from 1 mL of must or wine. The same DNA was used for DGGE, QPCR and PCR amplification of the ribosomal region. 2.4. PCR and restriction analysis 2.2. Wine fermentations and sampling This study was conducted in the experimental cellar of the Faculty of Oenology in Tarragona (Spain) during the 2007 vintage in semi-industrial conditions. Macabeo was the grape variety chosen for the vinifications. After destemming and crushing the grapes, SO2 was added (60 mg/L) and the must settled at 10 °C to separate the particles by density. Afterwards, the clear must was transferred Table 1 Reference strains used in this study. Sources of strains are abbreviated as: Spanish Type Culture Collection (CECT), Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSM) and Culture Collection of the Laboratorium voor Microbiologie, Gent (LMG). Lactic acid bacteria Lactobacillus brevis Lactobacillus buchneri Lactobacillus plantarum Leuconostoc mesenteroides Oenococcus oeni Pediococcus parvulus Pediococcus pentosaceus Acetic acid bacteria Acetobacter aceti Acetobacter oeni Acetobacter pasteurianus Gluconacetobacter hansenii Gluconobacter oxydans Yeast Candida boidinii Candida mesenterica Candida sake Candida stellata Dekkera anomala Hanseniaspora guilliermondii Hanseniaspora uvarum Issatchenkia terricola Saccharomyces cerevisiae Torulaspora delbrueckii Zygosaccharomyces rouxii to two 80-L tanks and fermented at 25 °C and at 13 °C. The fermentation temperature was continuously monitored and refrigerated by circulating cool water in a double-jacket stainless steel vat. Both fermentations were conducted by spontaneous microbiota (without yeast inoculation). After settling, the must had 180 g/L reducing sugar concentration, 4.8 g/L of total acidity (expressed as tartaric acid) and a pH of 3.2. The final ethanol concentrations were 10.3 and 10.5 for the wines fermented at 25 °C and 13 °C respectively. Samples were taken from the must and the settled must at the beginning of fermentation (density of 1080 g/L), middle fermentation (density of 1050 g/L), at the middle-late fermentation stage (density of 1020 g/L) and at the end of fermentation (density of 990 g/L). Must and settled must were common for both fermentations. Samples were taken after homogenization by pumping-over. Several dilutions of each sample were plated on YPD-agar medium. Fifty colonies from each fermentation point were randomly isolated and purified for further identification. CECT CECT CECT CECT CECT CECT CECT 4121 4111T 220 219 217T 813 4695 DSM 2002, DSM LMG 21952T DSM 3509, DSM LMG 1529, DSM DSM 2343, DSM CECT CECT CECT CECT CECT CECT CECT CECT CECT CECT CECT 3508 46617 5602 7145 10029 1025 10034 11109 11162T 11029T 11107 11139, CECT 11176T 1942NT 1880, CECT 10558 1230, CECT 1232 The ITS region and the 5.8S rRNA gene were amplified as described previously (Guillamón, Sabaté, Barrio, Cano, & Querol, 1998). All the amplifications were performed in a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). Five microliters of the ITS/5.8S rRNA gene amplified product were digested with the restriction endonuclease HinfI according to the supplier’s instructions. CfoI, DraI or HaeIII were also used for further identification when needed. All the restriction enzymes were from Roche Diagnostics GmBh (Mannheim, Germany). 2.5. Direct cloning of ribosomal fragment of yeasts The amplicons of the ITS region and the 5.8S rRNA gene, which used the DNA extracted from wine samples as template, were cloned using pGEMÒ-T Easy Vector (Promega Corporation, Madison, WI) according to the manufacturer’s protocol. Fifty transformed Escherichia coli colonies from each sample were purified and their plasmids isolated. Standard procedures for bacterial transformation and plasmid isolation from E. coli were performed (Sambrook, Frisch, & Maniatis, 1989). Five microlitres of the isolated plasmid were digested with the restriction endonuclease HaeIII (Roche Diagnostics GmBh, Mannheim, Germany) according to the supplier’s instructions. 2.6. DGGE The primers we used to amplify the specific ribosomal region of each microbial group were: U1GC/U2 (Meroth, Walter, Hertel, Brandt, & Hammes, 2003) for yeasts, L1GC/HDA2 (Meroth, Hammes, & Hertel, 2003) for LAB and WBAC1GC/WBAC2 (Lopez et al., 2003) for AAB. For lactic acid bacteria, a fragment of the 16S rRNA gene was amplified and separated by DGGE as described by Meroth, Walter, et al. (2003). For acetic acid bacteria, the PCR amplification was prepared as in Lopez et al. (2003) although the electrophoretic run was kept at a constant 170 V for 4 h at a constant temperature 775 I. Andorrà et al. / Food Research International 43 (2010) 773–779 1080 1060 density (g/L) of 60 °C in TAE buffer 0.5, and in a denaturing gradient from 30% to 60% of urea and formamide. For yeasts, amplification of the fragments and denaturing electrophoresis was performed according to Meroth, Walter, et al. (2003). All PCR amplifications were performed in a Gene Amp PCR System 2700 (Applied Biosystems, Fosters City, USA), using EcoTaq DNA Polimerase (Ecogen, Spain). The Dcode universal mutation detection system (Bio-Rad, Hercules, Calif.) was used to run the DGGE analysis. 1040 1020 1000 2.7. Sequencing 980 0 The DNA fragments from the DGGE gels were excised according to Omar and Ampe (2000). Each excised band was then transferred into 50 lL of sterile water and incubated overnight at 4 °C to allow diffusion of the DNA. One microliter of the eluted DNA was used for re-amplification with primers without the GC clamp. The PCR products were purified and sequenced by Macrogen Inc. facilities (Seoul, South Korea) using an ABI3730 XL automatic DNA sequencer. The primers ITS1 and ITS4 were used for sequencing the ribosomal region inserted in the pGEM plasmid. The BLAST search (Basic Alignment Search Tool, Internet address: http://www.ebi.ac.uk/blastall/nucleotide.html) was used to compare the sequences obtained with databases of the European Molecular Biology Laboratory (EMBL). We considered identification to be correct when gene sequences showed identities of 98% or higher. 2.8. QPCR In all cases QPCR was performed on an ABI Prism 5700 Sequence Detection System (Applied Biosystems). Power SyberGreen master mix was used according to the manufacturer’s instructions (Applied Biosystems, CA). An ABI PRISM 96 well optical plate was used for the reaction. This instrument automatically determined the CT. Yeast and bacteria quantification was performed by using the primers: YEASTF/YEASTR (Hierro et al., 2006) for total yeast, CESP-F/SCER-R for Saccharomyces and CESP-F/HUV-R for Hanseniaspora (Hierro, Esteve-Zarzoso, Mas, & Guillamón, 2007), WLAB1/WLAB2 for lactic acid bacteria (Neeley, 2005) and AQ1F/ AQ2R for acetic acid bacteria (González, 2006), as described by Andorrà et al. (2008). In the case of C. zemplinina, the new pair of primers, AF (50 -CTAGCATTGACCTCATATAGG-30 ) and 200R (50 GCATTCCCAAACAACTCGACTC-30 ), were designed from the D1/D2 domain of the 26S rRNA gene. AF primer is specific for C. zemplinina while the 200R primer is homologous to a conserved region for all the yeasts used for the alignment. Therefore, the specificity was determined by the AF primer. Standard curves were created by plotting the Ct (Cycle Threshold) values of the QPCR performed on dilution series of cells against the log input cells/mL. Samples and cultures for standard curves were analysed in triplicate. 3. Results Microbial populations (yeast, lactic acid bacteria and acetic acid bacteria) were monitored by qualitative (DGGE), semi-quantitative (direct cloning of amplified ribosomal DNA) and quantitative (realtime PCR) culture-independent techniques. In order to evaluate the effect of the temperature of fermentation on the dynamics and diversity of these populations, the same grape-must was divided into two tanks and fermented at 25 °C (optimum temperature) and 13 °C (restrictive temperature). Both fermentations proceeded spontaneously (non-inoculated) and the low-temperature fermentation took longer (double time) to complete (Fig. 1). 5 10 15 20 25 30 Days of fermentation Fig. 1. Evolution of the wine fermentations at 25 °C (—) and 13 °C (- -) measured as density (g/L). 3.1. Species detection by DGGE The application of this technique to the different samples taken throughout both wine fermentations allowed us to detect the species of filamentous fungi Aspergillus niger and Botriotinya fuckeliana (teleomorph of Botrytis cinerea), the species of yeasts C. zemplinina, Hanseniaspora uvarum and Saccharomyces cerevisiae, and the species of acetic acid bacteria Acetobacter aceti. No species of lactic acid bacteria were detected at any point of the fermentations. The correct identification of these species was confirmed by excision from the gel, purification and sequencing of the different bands. The distribution of these species in the different fermentation stages is shown in Table 2. Filamentous fungi were only detected in the grape-must and they quickly disappeared with the fermentation process. H. uvarum was also detected in the grapemust and at the beginning of the alcoholic fermentation. However, C. zemplinina and S. cerevisiae were detected in all the samples analysed. Likewise, the species A. aceti was also ubiquitous throughout the process. The fermentation temperature did not modify the diversity and presence of these species during the alcoholic ferTable 2 Microbial population analysed using DGGE during alcoholic fermentation at both temperatures (13 °C and 25 °C). A.: Aspergillus, B.: Botriotinya, C.: Candida, H.: Hanseniaspora, S.: Saccharomyces, Ac.: Acetobacter. 13 °C 25 °C Must A. niger B. fuckeliana C. zemplinina H. uvarum S. cerevisiae Ac. aceti Settled must A. niger B. fuckeliana C. zemplinina H. uvarum S. cerevisiae Ac. aceti Beginning C. zemplinina H. uvarum S. cerevisiae Ac. aceti C. zemplinina S. cerevisiae Middle C. zemplinina S. cerevisiae Ac. aceti C. zemplinina S. cerevisiae Ac. aceti Middle-late C. zemplinina S. cerevisiae Ac. aceti C. zemplinina S. cerevisiae Ac. aceti End C. zemplinina S. cerevisiae Ac. aceti C. zemplinina S. cerevisiae Ac. aceti Ac. aceti 776 I. Andorrà et al. / Food Research International 43 (2010) 773–779 mentation. The only difference can be attributed to a faster disappearance of H. uvarum in the control fermentation (25 °C). 3.2. Species identification by direct cloning of amplified ribosomal DNA The ribosomal region, which spans the 5.8S gene and the ITS region, was amplified by using as template the DNA directly extracted from must and wine samples. The PCR product was ligated into a plasmid and cloned in an E. coli strain. Fifty E. coli colonies per sample were analysed by plasmid purification and restriction analysis of this plasmid. We had cloned the same PCR product of C. zemplinina, H. uvarum and S. cerevisiae reference strains, as the major species found in the process, and obtained the restriction pattern of the plasmid with ribosomal region inserted. Only clones giving restriction patterns different from those of the major species were identified by sequencing. We also cultured the must and wine samples in a plate and the same number of yeast colonies were randomly analysed by amplification and restriction of the same ribosomal region (Esteve-Zarzoso, Belloch, Uruburu, & Querol, 1999; Guillamón et al., 1998). The percentage of the different yeast species detected throughout fermentations by both culture-independent and culture-dependent methods is shown in Table 3. Yeast diversity detected by direct cloning of the PCR product was higher than by the DGGE technique. Seven different species were identified: A. niger, C. zemplinina, H. uvarum, Hanseniaspora vineae, S. cerevisiae, Saccharomycopsis vini and Zygosaccharomyces bailii. However, the total of C. zemplinina and S. cerevisiae repre- sented 95% of the colonies analysed. Moreover, the remaining species were only isolated in the grape-must samples. Regarding the two major species, as the fermentation progressed the percentage of S. cerevisiae increased and C. zemplinina decreased. However, this latter species was still present to a significant degree at the end of the fermentation. Again, the fermentation temperature hardly influenced the species distribution. The same species and similar percentages were detected in both fermentations. It should be noted that the grape-must samples, which showed the highest diversity, were common for both fermentations. Of particular interest is the comparison of the direct cloning with the random identification of yeast colonies. The culture of the must and wine samples increased the percentages of the major species and decreased the number of species detected (only H. uvarum and H. osmophila were identified from the minor species). Furthermore, C. zemplinina showed an absolute predominance at the beginning of the process while all the colonies analysed at the end of the process belonged to S. cerevisiae. The only noteworthy differences between the yeast colonies identified in the fermentations at 13 °C and 25 °C are that C. zemplinina disappeared more rapidly at low temperature and H. osmophila was only isolated in the 25 °C fermentation. 3.3. Enumeration of yeast by real-time quantitative PCR (QPCR) We used QPCR to enumerate the following main wine yeast groups: total yeasts, Saccharomyces, Hanseniaspora and C. zemplinina. The pair of primers for the quantification of C. zemplinina was Table 3 Microbial population analysed using cloning and plating techniques during alcoholic fermentation at both temperatures (13 and 25 °C). Values indicate the% of the colonies analysed on each fermentation point. A.: Aspergillus, C.: Candida, H.: Hanseniaspora, S.: Saccharomyces, Smycopsis.: Saccharomycopsis, Z.: Zygosaccharomyces. Must Settled must Beginning Middle Middle-late End Plate Cloning – 2 – 2 – – – – – – – – C. zemplinina Plate Cloning 94 68 96 66 96 52 12 22 2 24 – 32 H. uvarum Plate Cloning – 6 4 4 4 8 2 – – – – H. vineae Plate Cloning – 2 – – – 2 – 4 – – – – S. cerevisiae Plate Cloning 6 16 – 28 – 38 86 74 98 76 100 68 Smycopsis. vini Plate Cloning – 4 – – – – – – – – – – Z. bailii Plate Cloning – 2 – – – – – – – – – – Plate Cloning – 2 – 2 – – – – – – – – C. zemplinina Plate Cloning 94 68 96 66 78 82 34 30 8 16 – 18 H. osmophila Plate Cloning – – – – 10 – – – – – – – H. uvarum Plate Cloning – 6 4 4 2 – – – – – – – H. vineae Plate Cloning – 2 – – – – – – – – – – S. cerevisiae Plate Cloning 6 16 – 28 10 18 66 70 92 84 100 82 Smycopsis. vini Plate Cloning – 4 – – – – – – – – – – Z. bailii Plate Cloning – 2 – – – – – – – – – – 13 °C A. niger 25 °C A. niger 777 I. Andorrà et al. / Food Research International 43 (2010) 773–779 3.4. Enumeration of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) The two main bacterial groups of wine were also counted by QPCR (Fig. 2), using the specific primers described for AAB (González, Hierro, Poblet, Mas, & Guillamón, 2006) and LAB (Neeley, Phister, & Mills, 2005). LAB population showed a very low count in the grape-must (102 cells/mL). This population ranged from 102 to 103 cells/mL throughout the process, regardless of the temperature of fermentation. The counts of AAB (approximately 5  105 cells/mL) were higher than LAB in the grape-must. The beginning of the fermentation produced a decrease in the AAB population of approximately 2 log units. These populations did not change during the fermentation, with the exception of the last day of fermentation at 13 °C. 4. Discussion Traditional methods of micro-organism quantification and identification rely on culturing the sample, counting and identifying colonies. These studies based on culture-dependent tools are likely to produce biased results based on unrepresentative cultivation conditions (Renouf, Strehaiano, & Lonvaud-Funel, 2007). Minor populations and stressed or weakened cells, which need specific Lactic and acetic acid bacteria (Cells ml-1) designed from the D1/D2 region of the 26S rDNA. Due to the high degree of phylogenetic relationship (and the low number of nucleotide substitutions) between the species C. stellata and C. zemplinina, it was impossible to design completely specific primers for one of these species. However, Sipiczki et al. (2005) showed that most of the wine strains preserved in culture collections or described in recent publications as C. stellata were indeed C. zemplinina. Likewise we also isolated C. zemplinina by DGGE and direct cloning but we never detected the presence of C. stellata. Therefore we can assume that these primers are useful to enumerate C. zemplinina in wines. The tests of specificity and sensitivity of this pair of primers were satisfactory (correlation coefficient 0.995, slope 3.207 and intercept 39.64) and comparable with the values obtained for the other primers (Hierro, Esteve-Zarzoso, Mas, & Guillamón, 2007; Hierro et al., 2006). A population size of approximately 107 cells/mL was quantified in the grape-must (Table 4). The settling of this must produced a decrease of approximately 50% (5  106 cells/mL) and the population grew to ca. 5  107 cells/mL during the fermentation. The maximum population size remained constant throughout the whole process at 13 °C whereas it decreased in the latter stages at 25 °C. C. zemplinina and Hanseniaspora were the major species in the grape-must, the number of Saccharomyces being much lower (1  104 cells/mL). At the beginning of fermentation, all the groups of species grew. The group with the highest counts was C. zemplinina (2  107 cells/mL) which represented the majority of the total population. Saccharomyces (1.8  106 cells/mL at 13 °C at 25 °C) and Hanseniaspora and 6.6  106 cells/mL (1.5  106 cells/mL) populations were one log unit lower. This latter species decreased its population size during the process to 105 cells/mL. The population of C. zemplinina did not increase after the first day of fermentation, but it was constant during the process and only decreased at the end of fermentation. The most important increase in yeast population was registered by the Saccharomyces group, which increased more than two orders of magnitude (or 2 log units) the first day and reached a maximum value of 1–2  107 cells/mL. However, the percentage of Saccharomyces barely reached 50% of the total population in the different days analysed, showing similar percentages to C. zemplinina. Also of interest was the fact that the total of the three groups of species analysed represented more than 80% of the whole population during the process, appearing once again as the major species of the wine-making process. However, these species only represented 33% of the total yeast population in the grape-must, indicating that other species are present, as we have detected with the other techniques used. 1,00E+06 1,00E+05 1,00E+04 1,00E+03 1,00E+02 1,00E+01 1,00E+00 0 5 10 15 20 25 30 Days of fermentation Fig. 2. Evolution of Acetic Acid Bacteria and Lactic Acid Bacteria analysed by QPCR during two fermentations: Lactic Acid Bacteria 25 °C (—N—) and 13 °C (- -D- -). Acetic Acid Bacteria 25 °C (—j—) and 13 °C (- -h- -). Table 4 Quantification (cells/mL) of yeast population by QPCR during fermentations at both temperatures (13 °C and 25 °C). Total yeast, Hanseniaspora, Candida stellata/zemplinina and Saccharomyces yeasts were evaluated. a Must Settled must Beginning Middle Middle-late End 13 °C Total yeast Candida Hanseniaspora Saccharomyces C + H + Sa 1.28 ± 0.41  107 1.82 ± 0.08  107 2.54 ± 0.85  106 1.10 ± 0.14  104 2.08  107 4.48 ± 1.91  106 9.05 ± 1.70  105 5.51 ± 0.22  105 8.49 ± 0.43  103 1.46  106 1.43 ± 0.34  107 2.08 ± 0.41  107 1.42 ± 0.13  106 1.83 ± 0.18  106 2.41  107 3.41 ± 0.92  107 1.18 ± 0.14  107 3.00 ± 1.79  105 1.63 ± 0.07  107 2.84  107 4.92 ± 0.19  107 1.75 ± 0.08  107 2.69 ± 0.62  105 1.19 ± 0.11  107 2.97  107 4.46 ± 2.16  107 6.62 ± 0.58  106 1.77 ± 0.22  105 9.82 ± 1.35  106 1.66  107 25 °C Total yeast Candida Hanseniaspora Saccharomyces C + H + Sa 1.28 ± 0.41  107 1.82 ± 0.08  107 2.54 ± 0.85  106 1.10 ± 0.14  104 2.08  107 4.48 ± 1.91  106 9.05 ± 1.70  105 5.51 ± 0.22  105 8.49 ± 0.43  103 1.46  106 3.29 ± 0.35  107 2.79 ± 0.39  107 1.48 ± 0.69  106 6.64 ± 0.25  106 3.6  107 3.55 ± 0.81  107 1.77 ± 0.66  107 1.40 ± 0.27  105 2.25 ± 0.25  107 4.03  107 4.35 ± 0.44  107 4.45 ± 0.62  107 1.62 ± 0.43  105 1.93 ± 0.22  107 6.39  107 1.70 ± 0.14  107 2.92 ± 1.20  106 1.29 ± 0.22  105 6.28 ± 0.27  106 9.33  106 Represents the addition of the yeast population of the Candida, Hanseniaspora and Saccharomyces yeasts. 778 I. Andorrà et al. / Food Research International 43 (2010) 773–779 culture conditions, may not be recovered on a plate. These limitations, associated with traditional culture-based methods, have driven microbiologists to develop alternative culture-independent techniques which are primarily based on the analysis of nucleic acids (Justé, Thomma, & Lievens, 2008). DGGE has been reported as a powerful technique for the study of the ecology of wine (Cocolin, Bisson, & Mills, 2000; Mills et al., 2002). However, as also reported in previous studies (Andorrà et al., 2008; Renouf et al., 2007), the main drawback of this technique is its low sensitivity. Minor species were barely detected, especially when the best adapted species constituted an overwhelming majority. Consequently, we were only able to detect a high diversity of minor species by DGGE in the grape-must samples in which no species showed a clear predominance. During wine fermentation the predominant species, C. zemplinina and S. cerevisiae, were the only species detected. We also used the direct cloning of an amplified ribosomal region of yeasts and the analysis of the clones by restriction analysis or sequencing. This technique detected higher yeast species diversity than DGGE and also permitted the calculation of the percentage or preponderance of the different species. However, it should be taken into account that only a small fraction of the population is being analysed and thus only semi-quantitative or qualitative conclusions should be drawn. Due to the simplicity of current cloning systems, this is an affordable, easy and reliable technique for the study of microbial diversity which avoids the problems associated with the cultivability of micro-organisms. Nevertheless, certain shortcomings may be attributed to this technique such as the presence of inhibitors in the matrix which interfere with the PCR reactions or differential efficiency in the DNA purification and amplification of the different species. In addition, inter-specific differences such as variation in the copy number of the ITS region may also produce biased results. Comparing the results of direct cloning with those obtained from yeast isolated colonies, the former also detected more species and the presence of predominant species did not prevent the detection of others present in low quantities. For example, from the middle to the end of the fermentation, few of the colonies analysed were identified as C. zemplinina whereas this species was detected in significant percentages in the same samples by direct cloning. The greater detection of this species by cloning may be explained by the inability of these species to grow under the culture conditions used or that with the direct cloning we are amplifying DNA from dead cells. This latter problem may be overcome by cloning cDNA instead of genomic DNA, since RNA is less stable than DNA after cellular death. The third technique used was the real-time quantitative PCR (QPCR). The DNA extracted from grape-must and wine samples was used with the specific primers and conditions designed in our previous works (Hierro et al., 2006, 2007), with the exception of the primers for the quantification of C. zemplinina, which were specifically designed for this study. In our opinion, the main advantage of this technique is that, regardless of the overwhelming presence of a major species, the specificity of the primers designed permits the detection of minor species. Thus, we were able to detect Saccharomyces in the grape-must samples and Hanseniaspora at the end of the fermentation, even though they each only represented less than 1% of the total population. This sensitivity is inconceivable with the other techniques used. However, there is a possible drawback of the technique such as we used it. The use as template of a very stable molecule such as DNA may inflate the counts by amplifying DNA from dead cells. We are currently assaying the use of a DNA-intercalating dye such as ethidium monoazide bromide, which penetrates only dead cells, avoiding its DNA amplification (Nocker & Camper, 2006). However, the correlation between quantification by plating (data not shown) and quantification by QPCR was satisfactory with some divergences. The total yeast counts of the must and settled-must samples were higher with QPCR than with plating (YPD-agar). As already reported Hierro et al. (2006), the total-yeast primers also amplify other fungal DNA; therefore, the presence of DNA from filamentous fungi, as detected by DGGE and cloning, would increase the values of the population. Our results clearly showed the significant loss of yeast population with the settling of the must (50% reduction). As previously reported (Torija, Rozès, Poblet, Guillamón, & Mas, 2001), Saccharomyces strains were the most competitive ones in the process, increasing their population by three orders of magnitude in a few days. However, in this case, the imposition of Saccharomyces was not absolute. C. zemplinina presented similar values to Saccharomyces throughout the fermentation, to the extent that it could be concluded that the grape-must was co-fermented by both groups of species. Regarding the evolution of bacteria population, LAB were practically unaffected by the process, with a constant low population from the beginning. AAB population size was considerably higher than LAB population in the must. The production of carbon dioxide by yeast during alcoholic fermentation substantially reduced the population of AAB. The idea that temperature may affect the ecology of wine fermentation has been previously reported. Sharf and Margalith (1983) suggested that H. uvarum had better ability than S. cerevisiae to grow at lower temperatures and Heard and Fleet (1988) showed that H. uvarum and C. stellata retained high populations until the end of fermentations at low temperatures. Likewise, Ribereau-Gayon et al. (2000) reported that the low temperature notably reduced the growth of acetic and lactic acid bacteria. However, in our fermentation conditions, the temperature of fermentation was not a determining factor in the yeast species development. The most notable differences are a rapid disappearance of Hanseniaspora at 25 °C and a high number of AAB at 13 °C, possibly as a consequence of the sluggish fermentation rate during the last days of fermentation at low temperature. In conclusion, DGGE and direct cloning of amplified DNA allowed us to detect higher species diversity compared with plating. Cloning was more sensitive in detecting minor species. The specificity of the primers designed for the QPCR allowed the enumeration of minor microbial groups in spite of the major presence of other species. The results of this study mostly confirmed the importance and distribution during the process of the major yeast species, widely reported in numerous studies. However, the use of these culture-independent techniques evidenced a higher presence and permanence of the non-Saccharomyces species and their contribution is not only limited to the first days of fermentation. Also noteworthy is the ubiquitous presence of a significant population of AAB throughout the process. Despite the anaerobic conditions established during fermentation, this population can survive under these circumstances (Bartowsky & Henschke, 2008; Millet & Lonvaud-Funel, 2000). It should be reminded that the fermentations proceeded spontaneously and the lack of inoculation prevented a rapid dominance of S. cerevisiae, the most powerful fermentative species. This circumstance might explain these high levels of AAB. Finally, in our working conditions, the temperature of fermentation showed a limited influence on the diversity and distribution of the different wine micro-organisms. Acknowledgements The present work has been financed by the Projects AGL200766417-C02-02/ALI and AGL2007-65498-C02-02/ALI of Spanish Ministerio de Educacion y Ciencia. The authors would also like to thank the Language Service of the Rovira i Virgili University for checking the manuscript. I. 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