Analytical composition of wines at final AF (stage 3) and final MLF (stage 6) at each vintage.
The microorganisms play an essential role in winemaking since a mixed culture of numerous microorganisms including fungal, yeast, and bacteria species are involved in this process and are the responsible for the final quality of the wine (Bisson et al., 1993). Therefore, in order to control the fermentation processes knowing and understanding the complex microbiota involved in them is necessary.
Yeast are able to convert sugar from grapes into ethanol and many other changes that lead to wine. Lactic acid bacteria (LAB) that are often present on the surface of the grapes and can represent significant populations in musts (Lonvaud-Funel, 1999) play dual roles in wine fermentations: as wine spoilage agents and as the main effectors of malolactic fermentation (MLF). Numerous studies have been conducted on the LAB that occur on grapes, grape musts and wines and it is generally agreed that a succession of species happens during the different stages of winemaking and conservation of wines (Ribéreau-Gayon et al., 2006). Most bacterial species present in wine fermentations have been identified by traditional microbiological techniques involving cultivation. However, as it was observed with microbial ecology studies of other environments, cultivation-dependent methods often exhibit biases resulting in an incomplete representation of the true present bacterial diversity (Amann et al., 1995; Hugenholtz et al., 1998). Applications of culture-independent molecular techniques to monitor the microbial successions of various food and beverage fermentations have revealed microbial constituents and microbial interactions not witnessed by previous plating analyses (Giraffa & Neviani, 2001).
Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis to separate bacterial 16S ribosomal DNA (rDNA) amplicons are common culture independent methods employed to characterize microbial communities from specific environmental niches (Lopez et al., 2003; Muyzer & Smalla, 1998). These approaches are attractive since they enable to detect individual species as well as to get overall profiling of community structure changes with time.
Otherwise, ecology, interactions and development of the different bacterial strains during alcoholic fermentation (AF) and MLF are still a field of active research. Efficient and precise methods of strain identification and discrimination have been developed during the last years, either to prepare well-defined starters of biotechnological interest in winemaking, or to quickly assess the presence of certain strains in a wine, or to gain insight of such a complex ecosystem as wine.
Pulsed field gel electrophoresis (PFGE) has proved to be an useful tool for the identification of a wide LAB strains variety and especially for species belonging to the genus
For all these reasons, the aims of this work were: (a) applying the DGGE, PFGE and RAPD-PCR techniques to the analysis of the LAB species diversity and the intraspecific diversity of
2.1. Wine production and wine samples
Traditional red wine fermentations from c.v. Tempranillo local grapes of 2006, 2007 and 2008 vintages at one winery of the Spanish northern region of Rioja were studied. Winemaking practices were the typical of this wine-producing area: AFs were conducted in the presence of grape skins, seeds and stalks, after the addition of sulphur dioxide and until the residual reducing sugar content was under 2 g/L. At this final point of AF, wines were drawn off into tanks and were allowed to undergo spontaneous MLF with the endogenous microbiota. The sampled winery had never used commercial starters for MLF. One fermentation tank was sampled in each vintage. Wine samples were collected aseptically for chemical and microbiological analysis at different times: must (stage 1), tumultuous AF (density around 1,025; stage 2), at final AF (< 2 g/L glucose + fructose; stage 3), initial MLF (consumption of 10% of the initial malic acid; stage 4), tumultuous MLF (consumption of 60% of the initial malic acid; stage 5) and at final MLF (L-malic acid concentration < 0.5 g/L; stage 6).
Oenococcus oenistarter samples
Sixteen commercial starter cultures employed to induce MLF derived from six different companies were analyzed. These commercial cultures were selected between the most frequently used in Spain.
2.3. Chemical analysis of the musts and wines
Alcohol degree, pH, total acidity, volatile acidity, reducing sugars, free and total sulphur dioxide and L-malic and L-lactic acid content were measured according to the European Community Official Methods (European Community, 1990).
2.4. Culture dependent methods
2.4.1. Bacterial enumeration and isolation
Must or wine samples were diluted in sterile saline (0.9% NaCl) solution and plated on modified MRS agar (Scharlau Chemie S.A., Barcelona, Spain) plates supplemented with tomato juice (10% v/v), fructose (6 g/L), cysteine-HCl (0.5 g/L), D,L-malic acid (5 g/L) and pymaricine (50 mg/L) (Acofarma, S. Coop., Terrassa, Spain). Samples were incubated at 30 ºC under strict anaerobic conditions (Gas Pak System, Oxoid Ltd., Basingstoke, England) for at least ten days, and viable counts were reported as the number of CFU/mL. Fifteen colonies from each wine sample were selected for reisolation and identification. Isolates were stored in 20% sterile skim milk (Difco) at –20 ºC.
Every commercial lyophilized starter culture was hydrated in saline solution (0.9% NaCl) and then 100 μL aliquot from the appropriate dilution was plated at the surface of modified MRS agar without pymaricine. Because of their low viability in laboratory conditions (Maicas et al., 1999a; Maicas et al., 1999b) glycine (40 mM) and ethanol (10% v/v) were added to this medium. The plates were incubated for at least 10 days at 30 ºC under anaerobic atmosphere (Gas Pak System, Oxoid Ltd.) and five colonies were isolated from each one.
2.4.2. Species identification
Species identification was carried out by previously recommended methods, which included bacteria morphology, Gram staining, and catalase (Holt et al., 1994).
2.4.3. Oenococcus oeni typification by PFGE
PFGE was carried out according to the method described by Birren et al. (1993) with some modifications (Lopez et al., 2007) for agarose block preparation. Because of the difficulty of typing commercial strains first of all these cells underwent to fifteen minutes of ultrasounds, moreover a higher quantity of lysozime (100 μL/block) was added and incubated for 2 h. Macrorestriction analysis was performed with two endonucleases:
2.4.4. Oenococcus oeni typification by RAPD-PCR
RAPD-PCR was carried out following the procedure described by Ruiz et al. (2010b) with some modifications: MgCl 100 mM, dNTP 50 mM and primer M13 100 mM. RAPD-PCR reaction was developed in a total volume of 50 μL and it was carried out with a Perkin Elmer, GeneAmp PCR System 2400 thermocycler. 20 μL of amplified products were resolved by electrophoresis in a 1.4% agarose gel in 0.5x TBE (45 mM Tris base, 89 mM, boric acid, 2.5 mM EDTA pH 8) for 3 h at 70 V.
2.4.5. Numerical analysis of PFGE and RAPD-PCR images
The conversion, normalization and further processing of images were carried out by InfoQuestTM FP software version 5.10 (Bio-Rad, USA). Comparison of the obtained PFGE patterns was performed with Pearson’s product-moment correlation coefficient and the Unweighted Pair Group Method using Arithmetic averages (UPGMA). Comparison of the pulse types from the PFGE and RAPD was made by composite data set comparison with average molecular analysis by Unweighted Pair Group Method using Arithmetic averages (UPGMA) (Ruiz et al., 2008).
2.5. Culture independent methods
2.5.1. Direct DNA extraction from wines samples
A volume of 10 mL of each must or wine sample was centrifuged (30 min, 10000xg, 4 ºC). The supernatant was discarded and 1.2 mL of saline solution (NaCl 0.9%) and 2.4 mL of zirconium hydroxide (7 g/L) were added to the pellet to facilitate pelleting of the bacteria in wine (Lucore et al., 2000). After horizontal shaking during 10 min at room temperature, the suspension was again centrifuged (10 min, 500xg, 7 ºC) and finally DNA was purified from the cell pellet by using a PowerSoil® DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA USA) as per the manufacturer’s instructions.
2.5.2. PCR conditions
PCR was performed using an Applied Biosystem, GeneAmp® PCR System 2700 thermocycler at a final volume of 50 µL. To amplify the region V4 to V5 of 16S rDNA gene, primers WLAB1 and WLAB2GC were used as López et al. described (2003). Moreover, primers
2.5.3. PCR-DGGE analysis
The separation of the respective PCR products was performed with the D-CODETM universal mutation detection system (Bio-Rad, Hercules, Calif.). PCR products obtained from WLAB1-WLAB2GC primers were run on 8% (wt/V) polyacrilamide gels in a running buffer containing 2 M Tris base, 1 M Glacial acetic acid and 50 mM EDTA pH 8 (TAE), and a denaturing gradient from 35 to 55% of urea and formamide. The electrophoresis was performed at 20 V for 10 min, and 80 V for 18 h at a constant temperature of 60 C. PCR products generated with the
2.5.4. DNA sequencing and phylogenetic analysis
PCR products were sequenced by Macrogen Inc. (Seoul, South Korea). The quality and characteristics of the obtained sequences were analyzed with the software InfoQuestTM FP 5.10, only those ones considered as appropriate were used for comparison to the GenBank database with the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). After this preliminary study, our sequences and their homologous ones (obtained from the Nucleotide Database: http://www.ncbi.nlm.nih.gov/nuccore) were assembled and submitted to phylogenetic and evolutionary analysis with MEGA version 4.0.2 (Tamura et al., 2007). The Neighbor-Joining analysis (Saitou & Nei, 1987) allowed to get information about the relations between the gotten sequences and the reliability of the identifications provided by the Nucleotide Database. The bootstrap test was based on 1000 replicates (Felsenstein, 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2007) that allowed to calculate the equivalent units to the base substitutions per site.
3. Results and discussion
3.1. Oenological parameters of wine samples and fermentation development
Results for analytical composition of wines during three vintages are displayed in Table 1. Data were within the usual range of Tempranillo wines from this Spanish region (González-Arenzana et al., 2012b). After completion AF, alcohol content ranged between 13.0% and 14.0%, pH was between 3.32 and 3.64 and free SO2 level was between 4.24 and 18.1 mg/L. During MLF a decrease in total acidity and a subsequent increase in pH were observed. In addition, an increase in volatile acidity was noted as it was expected. The wine from 2006 vintage showed less restrictive parameters for microbial growth, so it presented higher pH and the lowest values of alcohol content and SO2.
|Alcohol content (% v/v)||13.0||-||13.8||-||14.0||-|
|Total acidity (g/L tartaric acid)||7.98||5.91||7.63||6.71||9.00||7.20|
|Volatile acidity (g/L acetic acid)||0.25||0.46||0.37||0.49||0.26||0.37|
|Total SO2 (mg/L)||28.4||-||38.1||-||31.6||-|
|Free SO2 (mg/L)||4.24||-||18.1||-||13.2||-|
|L-malic acid (g/L)||2.60||0.04||1.48||0.16||2.61||0.21|
|L-lactic acid (g/L)||-||1.81||-||1.10||-||1.72|
AF completion lasted for six, sixteen and eleven days in 2006, 2007 and 2008 vintages, respectively. Viable LAB counts during AF were in the range of 102 - 103 CFU/mL, increasing to 107 – 108 CFU/mL during MLF, similar to spontaneous MLF results reported by other authors (European Community, 1990; Lopez et al., 2008). The development of the MLF was related to the viable population of LAB and there was a relation between bacterial population and decrease in L-malic acid (data not shown). Important differences in MLF duration were observed between vintages and MLF completion lasted for 21, 239 and 136 days in 2006, 2007 and 2008 vintages, respectively. Different temperatures at each vintage (wine temperature below 12 ºC after AF in 2007) and the lack of temperature control in the winery were the determinant factors in these differences, but factors such as pH, composition of the wine and the interaction with other microorganisms implicated in the fermentation could also influence, as it has been reported by other authors (du Plessis et al., 2004; Lonvaud-Funel, 1999; Reguant et al., 2005a; Reguant et al., 2005b).
3.2. Species identification
3.2.1. Culture dependent microbiological analysis
Figure 1 shows the number of isolates of the viable LAB species identified at each stage and year of vinification. A total of 251 LAB isolates were recovered and identified as belonging to eight different species. The greatest diversity of LAB species was detected during the AF.
3.2.2. Culture independent microbiological analysis and comparison with culture dependent method
PCR-DGGE analysis of the sampled wine fermentations in the three studied vintages using primers WLAB1/2 (16S rDNA-based primer sets) and primers
The sequences obtained from the DNA excised DGGE-bands of each sample and their homologous ones from Nucleotide Database (Altschul et al., 1990) constituted a tree for each studied gene (Figure 3). Figure 3a shows a tree based on 16S rDNA gene composed by four ramifications or branches belonging to the genus
The species identification at each stage of vinification in the three studied years with culture independent techniques (16S rDNA/PCR-DGGE and
A total of fourteen different LAB species were identified in the three studied vintages by traditional and culture independent methods. PCR-DGGE analysis allowed to identify nine species in comparison to the eight ones detected by culture in plate of the sampled wine. Thus,
Results about diversity of LAB species found at each year and stage of vinification were very similar to those described above for culture dependent method. The greatest diversity was detected again during AF, opposite to MLF were only two species were present,
|Detected LAB species||2006||2007||2008|
|Total species nº||7||3||4||1||1||1||6||3||2||2||1||1||3||6||3||1||1||1|
|Plate species nº||4||2||2||1||1||1||5||2||1||1||1||1||2||3||1||1||1||1|
|Total DGGE species nº||4||1||2||1||1||1||2||1||2||2||1||1||2||4||3||1||1||1|
|16S rDNA/DGGE species nº||4||1||2||1||1||1||1||1||2||2||1||1||2||3||3||1||1||1|
3.3. Strain typing of
Oenococcus oeni 3.3.1. PFGE analysis of the strains of this study
Identification of the
Comparing coincident genotypes for the three vintages, it was observed that between the genotypes isolated in 2006 vintage two were found in 2007 (genotypes 18 and 20) and four in 2008 (genotypes 3, 13, 17 and 18). Moreover, two genotypes isolated in 2007 (genotypes 18 and 25) were also detected in 2008 vintage. Only one genotype (18) was identified in the three studied years. The frequency of participation of each genotype varied from year to year, thus dominant genotypes one year were minority or not present at other one which suggested the adaptation of
Interestingly, no genotype was isolated in all fermentation stages so fourteen genotypes appeared only at AF (stages 1-3), six were present at all MLF stages (4, 5 and 6) and three of them were also detected at the end of AF. Most fermentation stages showed mixed
3.3.2. Comparison of the PFGE and RAPD-PCR profiles from the wine fermentation strains and commercial strains
The thirty-seven genotypes of the indigenous O
These all four autochthonous genotypes were detected in 2006 vintage, year which showed the greatest strain diversity, two of them occurred in 2008 and one in 2007, with an important frequency of appearance in some cases (Table 3).
|Genotype||Isolation stage||Frequency of appearance at each vintage (%)|
Therefore, despite two of these strains had been previously considered as interesting for the selection of new malolactic starter cultures, the possible identical strain identification with already marketed strains suggested reject these two indigenous
This study has been a contribution to a better description of the LAB ecology along the process of Tempranillo wines winemaking.
The study about the microbial diversity of viable LAB populations showed that the species diversity was higher at the AF stage where eight different species were identified.
This work allowed to increase the endogenous strain collection of LAB isolated from fermenting wines of the Appellation of Origin Rioja what meant a contribution to the preservation of biodiversity and wine peculiarity of this region and a starting point for future research.
The analysis of the total LAB populations by culture independent techniques (PCR-DGGE) showed that the species diversity detected along the winemaking process was higher than the one found by the study of viable LAB, identifying up to nine different LAB species. The LAB species variability was also higher at the previous stages to the MLF. Once spontaneous MLF started this variability was greatly reduced, with
The results obtained with culture dependent and independent techniques were complementary so in studies conducted in microbial ecology they both should be used to achieve a broader view of the studied ecosystem.
PFGE has shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. The results of
Several genotypes could be considered as interesting
This work was supported by funding and predoctoral grant (B.O.R. 6th March, 2009) of the Government of La Rioja, the I.N.I.A. project RTA2007-00104-00-00 and FEDER of the European Community and was made possible by the collaborating winery.