Effect of malolactic fermentation by Pediococcus damnosus on the composition and sensory profile of Albariño and Caiño white wines

Authors


Abstract

Aim

This study was aimed to investigate the influence of malolactic fermentation (MLF) on sensory profile and organoleptic characteristics of Albariño and Caiño white wines.

Methods and Results

Autochthonous bacteria were isolated from wines after alcoholic fermentation (AF) and further identified as Pediococcus damnosus by 16S rRNA gene sequence analysis. When a commercial Oenococcus oeni starter was inoculated into Albariño and Caiño white wines to perform MLF, which was checked by HPLC quantification of malic and lactic acids, it was shown that autochthonous Ped. damnosus strains were able to predominate over the commercial O. oeni starter and perform MLF in Caiño wine. By contrast, neither commercial strain nor indigenous Pediococcus carried out MLF in Albariño wine. However, MLF was achieved when autochthonous strains that predominated in Caiño were inoculated into Albariño. Sensory analysis showed that after the MLF Albariño increased its body and softness, while Caiño result a more mature wine.

Conclusions

MLF can positively affect Albariño and Caiño wines giving them new attributes. Pediococci isolated and characterized in this work can successfully perform MLF without negative effects on the wine, because no production of biogenic amines or exopolysaccharides by the selected pediococcus strains was detected.

Significance and Impact of the Study

The effect of MLF in the sensory profile of Albariño and Caiño wines has never been studied before. Results obtained in this work showed that Ped. damnosus strains can be considered as a new topic of investigation on malolactic starter.

Introduction

The grape cultivar ‘Albariño’ is one of the oldest in the vine-growing area of Galicia in northwest Spain (Huetz De Lemps 1967). Until recently, this typical white grape variety was grown only for personal consumption, but nowadays, its area of production has increased enormously, being now one of the most prestigious grapes in Spain. Albariño wines are young balanced wines, fresh (due to a high acid content), and with an intense fruity and floral aroma (Dieguez et al. 2003; Oliveira et al. 2008; Armada et al. 2010).

The ‘Caíño’ cultivar group is considered to be the oldest grape variety in Galicia. Different types of ‘Caíño’ were first reported between 1909 and 1911 by García de los Salmones (1914). ‘Caíño Blanco’ is cultivated in the Rosal area, and it is used, like the Albariño, in the production of wines of recognized quality [Origin Denomination (OD) Rías Consejo Regulador De La Denominación De Origen Rías Baixas 2004; Santiago et al. 2007].

The aroma is a very important component of the organoleptic quality of wine. The profile is influenced by different factors, environmental and management practices (Jackson and Lombard 1993), grape varieties (Schreier et al. 1976; Gunata et al. 1985), winemaking techniques (Dubourdieu 1986) and yeasts (Soumalainen and Lehtonen 1979; Carrascosa et al. 2012) among others.

Winemaking normally involves two fermentation processes, alcoholic fermentation (AF) by yeast, principally Saccharomyces cerevisiae, and a second fermentation, called malolactic fermentation (MLF), which can be performed by different lactic acid bacteria (LAB), generally associated with three genera: Oenococcus, Lactobacillus and Pediococcus (Maicas et al. 1999).

MLF, which is conducted in most red and in some white wines, is not usually accomplished in Albariño wines, characterized by a typical fresh taste. The activity of the LAB that perform MLF decreases wine acidity through the conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid. MLF produces a number of compounds that modify flavour, being diacetyl one of the most recognized. Other compounds produced by LAB metabolism are 1-hexanol, ethyl acetate, ethyl lactate, diethyl succinate, γ-butyrolactone, glycoaldehye, glyoxal, 2,3-butanediol, and caprylic acid. The synthesis of these aromatic compounds promotes the reduction of vegetable and herbaceous aromas and the appearance of other fruity and floral aromas in wines (Sauvageot and Vivier 1997).

To obtain a more complex aroma, and also for the microbiological stabilization of wine, MLF is also conducted in some white wines. Moreover, LAB seems to have glycosidase activity, and they are able to release terpenes, norisoprenoids, phenols and vanillin from grape glycosidic precursors (Hernandez-Orte et al. 2009).

In most cases, Oenococcus oeni is the only species identified when the MLF is completed (Lonvaud-Funel et al. 1991), as it is the best adapted LAB to the difficult growing conditions (low pH, high ethanol content, and the presence of SO2) that characterize the wine. Oenococcus oeni is present in wine during AF together with other LAB belonging to Pedioccocus, Leuconostoc and Lactobacillus genera but, generally, towards the end of the AF, Lactobacillus, Pediococcus and Leuconostoc species progressively disappear (Lonvaud-Funel 1999).

Lactobacillus and Pediococcus are generally considered spoilage bacteria in wine, because some strains can synthesize exopolysaccharides and, consequently, provide a viscous and thick texture to the wine (Walling et al. 2005). Moreover, they can also produce high concentrations of acetic acid (Lafon-Lafourcade et al. 1983) and biogenic amines (BA) (Landete et al. 2005).

However, as previously suggested (Fugelsang and Edwards 2007), and as these negative properties are strain-related, the growth of lactobacilli and pediococci in wine may not necessarily affect adversely its quality but, on the contrary, may add desirable flavours and aromas under certain circumstances. In fact, because Lactobacillus and Pediococcus species possess a high number of enzyme encoding genes important for the production of wine aroma compounds, their use as starter cultures to carry on MLF has recently been investigated (Du Toit 2011; Strickland 2012).

This work reports the first study on the influence of MLF on sensory profile and organoleptic characteristics of Albariño and Caiño wines. Moreover, the native LAB strains of these white wines were investigated. Our study showed that MLF was accomplished by two indigenous Pediococcus damnosus strains that predominate over the inoculated O. oeni strain and that they did not present negative properties eventually related with the Pediococcus genus. Chemical and physical parameters and sensory analysis were performed before and after MLF to examine and evaluate the effect of this process on the sensory attributes of the wines. Results obtained show that the strains of Ped. damnosus isolated here are suitable starters for MLF in white wines and that MLF can positively affect Albariño and Caiño wines.

Materials and methods

Wine samples

A total of 12 L of Caiño and Albariño wines (vintage 2009) fermented in a winery located in the zone of the O Rosal in the province of Pontevedra in Galicia (Spain), pertaining to the Rías Baixas O.D., was used in this study. Samples were taken before and after MLF, and no SO2 was added during the process.

Wine composition analysis

Wines were analysed both before and after the MLF for the following chemical parameters: alcohol content, total dried extract, pH and total and free volatile SO2, according to official methods (G.U.C.E. n. 272 3/10/1990). The colour of wine was also tested using CIE-CIELAB parameters. Fixed acids were analysed by HPLC according to Cane (1990). Phenol composition (total polyphenols and flavonoids reaction to p-DACA) was determined according to Di Stefano et al. (1989). Physical and chemical composition of Albariño and Caiño wines, before (initial wine) and after MLF, is shown in Table 1.

Table 1. Physical and chemical parameters of wines before and after MLF
 Albariño before MLFAlbariño after MLFCaiño before MLFCaiño after MLF
  1. MLF, malolactic fermentation.

Current assays
Alcohol (%)12·98 ± 0·0413 ± 0·0312·46 ± 0·0212·46 ± 0·01
pH3·51 ± 0·003·68 ± 0·003·71 ± 0·003·79 ± 0·01
Total acidity (g/l)7·4 ± 0·005·2 ± 0·006·65 ± 0·075·00 ± 0·00
Volatile acidity (g/l)0·49 ± 0·030·525 ± 0·020·36 ± 0·010·41 ± 0·01
Free SO2 (mg/l)6·40 ± 0·004·8 ± 0·006·24 ± 0·223·40 ± 0·85
Total SO2 (mg/l)42·0 ± 4·2441·0 ± 1·4140·0 ± 1·4137·9 ± 2·83
Colour
E 420 nm0·17 ± 4·1 × 10−30·19 ± 2·1 × 10−40·11 ± 7·07 × 10−50·16 ± 3·54 × 10−4
CIE
Brightness Y%0·94 ± 4·36 × 10−30·93 ± 8·37 × 10−50·96 ± 1·5 × 10−30·92 ± 1·46 × 10−3
Saturation S%11·71 ± 0·1613 ± 0·017·74 ± 0·069·78 ± 3·67 × 10−3
CIELAB
L*97·66 ± 0·1797·31 ± 3·4 × 10−398·56 ± 0·0696·94 ± 2·62 × 10−4
a*−2·75 ± 3·04 × 10−3−2·44 ± 2·4 × 10−3−2·15 ± 0·03−1·97 ± 0·06
b*12·71 ± 0·1513·95 ± 0·018·57 ± 0·0710·51 ± 0·01
h*−1·36 ± 2·23 × 10−3−1·40 ± 3·1 × 10−4−1·33 ± 1·9 × 10−3−1·38 ± 0·01
C*13·00 ± 0·1414·17 ± 0·018·83 ± 0·0810·69 ± 0·02
Phenolic composition (mg/l)
Total phenolic content107·00 ± 5·65106·00 ± 1·4197·50 ± 0·7096·50 ± 3·54
Flavonoids reaction to p-DACA65·00 ± 1·4163·00 ± 1·4154·50 ± 0·7052·00 ± 1·41

Malolactic fermentation assays

To study the effect of MLF on these wines, they were inoculated with a commercial O. oeni starter (Lallemand, Italy) normally used in the winery. This dried commercial strain was rehydrated for 20 min in distilled water at 30°C and employed following manufacturer's instructions and then inoculated in 5 l of either Albariño or Caiño wines.

As no MLF was observed in Albariño wine, a second inoculation (106 cell ml−1) was performed with a mix of two indigenous strains of Ped. damnosus isolated from Caiño wine that had been previously grown in a wine-like medium (MRS diluted 1 : 10 with water and added with 3 g l−1 malic acid and 12% ethanol). Wines were incubated at 25°C until consumption of malic acid, which was quantified by HPLC (Waters Binary HPLC 1525 pump, Turin, Italy) using a RP 18 5 μm column (LiChrospher® 100; Merck KGaA, Darmstadt, Germany) as previously described (Cane 1990). External standards were purchased from VWR (Milan, Italy).

To confirm the results obtained from these fermentations, new tests were made by utilizing Caiño wine previously sterilized by filtration with 0·2-μm filter. Three tests were performed, in duplicate, with the selected Pediococcus and the commercial Oenococcus strain: (i) The inoculum for MLF was made with Ped. damnosus mix, (ii) The inoculum for MLF was made with O. oeni, and (iii) The inoculum for MLF was made with Ped. damnosus mix plus O. oeni. The level of inoculum was 106 cell ml−1 for both species. Malic acid consumption was monitored by HPLC as described previously.

Isolation and characterization of the LAB

Microbial strains and growth conditions

Samples of Caiño and Albariño wines, taken before and after MLF, were serially diluted in 0·9% (w/v) NaCl and 1 ml aliquots were plated on MRS agar, pH 4·8, (VWR) with 0·1 mg ml−1 of cycloheximide (Sigma-Aldrich, Milan, Italy) to suppress yeast growth. After anaerobical growth at 30°C, 50 colonies were randomly selected and grown individually in liquid MRS, pH 4·8, supplemented with 10% (v/v) tomato juice (Difco, BD, Milan, Italy), and further incubated at 30°C. LAB cultures were maintained at −80°C in the isolation medium supplemented with glycerol 50% (v/v).

DNA extraction

DNA was extracted with ‘Archive pure DNA, Yeast and Gram+ kit’ (5-PRIME, Hamburg, Germany). Cultures were centrifuged (15 000 g, 1 min), and the pellet was treated according to the kit manufacturer's instructions. DNA samples were measured in a spectrophotometer (BECKMAN COULTER DU® 730, Life Science UV/VIS, spectrophotometer, Beckman Coulter, Milan, Italy) at 260–280 nm.

Species identification

DNA was amplified with universal primers 63f-1387r (Marchesi et al. 1998) and sequenced (BMR-genomics, Padova, Italy). Sequence similarity searches were performed using the Blast algorithm (Altschul et al. 1990) at NCBI (www.ncbi.nlm.nih.gov) on the GeneBank databases and the Ribosomal Database Project (Cole et al. 2009).

Strain typing

RAPD-PCR was performed with primers Coc and On2 (Cocconcelli et al. 1995; Reguant and Bordons 2003) at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 40°C for 1 min and 72°C for 2 min, with a final extension step of 10 min. Amplified products were visualized by ethidium bromide staining after gel electrophoresis. The marker used was 1Kb DNA Ladder (Sigma, Milan, Italy).

The band pattern obtained were compared using Bionumerics (Applied Maths, Ghent, Belgium) Dice's similarity coefficients were calculated as 2n/a+b, where n = the number of matching bands and a + b = the total number of bands (matching and no matching), and strains were grouped by using the unweighted pair group method with arithmetic averages (UPGMA); the profile of the commercial strain inoculated was used for comparison.

Determination of the biogenic amines-producing capability

The presence of decarboxylase genes hdc, tdc and odc was assessed by PCR according to Costantini et al. (2006), and the capability to produce histamine, putrescine and tyramine was determined by TLC according to Garcia-Moruno et al. (2005).

Evaluation of the capability to produce exopolysaccharides

PCR assay was performed according to Walling et al. (2005) using primers PF1/PF8 that amplify the dps gene, which product is responsible for the glucan biosynthesis.

Sensory evaluation

The wines were evaluated before and after the MLF in two replicates by a CRA-ENO trained panel composed by 14 experts (seven male and seven female) aged 25–60 years in an ISO (8589-2007) tasting room. The procedure followed derived from the ISO standards (11035-1994). Firstly terms, describing colour, odour and flavour (taste and mouth-feel sensations) of the wines were collected. Afterwards, the intensity of the chosen descriptors was measured using an unstructured intensity scale ranging from 0 to 80 mm presented on a wheel. Wine samples (30–40 ml) at 10–12°C were poured in ISO (3591-1977) approved glasses immediately before analysis and covered by Petri dishes. Samples were presented in a random order with a three-digit code.

Data were analysed by two-way analysis of variance (anova) using XL Stat software (Addinsoft, Milan, Italy). Mean comparisons were performed by Tukey's test at < 0·05. The data processed were represented by the common descriptors of the wines before and after MLF and the repetition of the sessions. For both wines, Albariño and Caiño, the anova analysis made on the replications of the sensory session showed that there were not significant differences, and thus, the average of the data of the two replicates were considered.

Results

Development of MLF

Wines after AF were not sterilized to simulate the real conditions of the winery and were inoculated with an O. oeni commercial starter (about 106 cell ml−1) as described in material and methods; the MLF was considered concluded when the malic acid was totally consumed. The commercial starter was chosen because it is a strain currently utilized in wine industry and, according to the manufacturer, suitable for white wine because it points out fruity aromas.

In Caiño wine, the bacterial population after the AF, before the inoculation of the starter, was 4 × 102 cell ml−1 and was constituted by Ped. damnosus, as shown after 16S rRNA gene sequence analysis. In this wine, MLF was completed in 30 days (Fig. 1). To verify whether the inoculated Ooeni commercial starter was responsible for the MLF, bacteria present in the wine at the end of MLF (2 × 105 cell ml−1) were isolated and characterized. Results showed that O. oeni commercial strain did not conduct the MLF, as 16S rRNA gene sequence analysis showed that only pediococci, identified as Peddamnosus with an identity of 99%, were isolated. RAPD PCR typing of the strains showed two different band patterns. Comparison of the band profiles showed that the two biotypes present at the end of MLF were also present in the initial wine (Fig. 2). Therefore, these indigenous strains were able to predominate over the commercial O. oeni bacterial starter. Two strains, C5 and C8, were chosen as representative of the two identified biotypes.

Figure 1.

Consumption of malic acid and formation of lactic acid during malolactic fermentation in Caiño wine. Inoculation of commercial Oenococcus oeni starter at t = 0. (image_n/jam12392-gra-0001.png) malic ac; (image_n/jam12392-gra-0002.png) lactic ac.

Figure 2.

Dendrogram of patterns of Pediococcus damnosus isolated from Caiño wine after malolactic fermentation (MLF) showing the presence of two different biotypes. MLD OENOS, commercial strain. C5 and C8, autochthonous Ped. damnosus strains chosen as representative of each biotype. FC, bacteria isolated at the end of the MLF.

In Albariño wine, bacterial indigenous population after AF was 50 cell ml−1, and it was constituted by Ped. damnosus. MLF using commercial starter was not initiated after more than one month of incubation; therefore neither the O. oeni starter, nor the indigenous population were capable to predominate and start MLF. Wine was then inoculated (at the 45th day) with a mix of the two autochthonous Pediococcus strains isolated in Caiño wine (C5 and C8). Under these conditions, MLF was also finished in Albariño wine in less than 30 days after inoculation (Fig. 3). The band profiles of the colonies isolated at the end of MLF, obtained by RAPD PCR analysis, showed that they were grouped to only one of the two strains inoculated, the strain C5 (Fig. 4).

Figure 3.

Consumption of malic acid and formation of lactic acid during malolactic fermentation in Albariño wine. Inoculation of commercial Oenococcus oeni starter at t = 0. Inoculation of autochthonous Pediococcus damnosus mix (C5 and C8) isolated from Caiño wine at t = 45. (image_n/jam12392-gra-0001.png) malic ac; (image_n/jam12392-gra-0002.png) lactic ac.

Figure 4.

Dendrogram of patterns of Pediococcus damnosus isolated from Albariño wine after malolactic fermentation (MLF) (indicated as FALB). MLD, commercial Oenococcus oeni strain. C5 and C8, indigenous Ped. damnosus strains isolated from Caiño wine that was inoculated in Albariño wine. FALB, bacteria isolated at the end of the MLF.

These results confirmed that the autochthonous strains isolated in Caiño wine were well adapted to the production conditions of both Albariño and Caiño wines, particularly C5 strain that seems to be better adapted than C8 to the conditions of Albariño wine.

Further tests were made on Caiño wine that had been sterilized and inoculated as described in Materials and methods. After 35 days, MLF was only completed in those tests in which inoculation was performed with Pediococcus alone or with Pediococcus plus Oenococcus, respectively, while no MLF was observed in those only inoculated with Oenococcus. These results confirmed that Pediococcus is better adapted to these white wines. Moreover, these data showed that the failure of O. oeni to perform MLF in the wines is not due to a competition with the autochthonous Pediococcus, as in these test, the wine had been previously sterilized.

The main concern related with some Pediococcus strains is the production of glucan, an exopolysaccharide that causes an alteration of the wine called ‘ropiness’; therefore, to evaluate the possible utility of these autochthonous strains as starters for MLF, the presence of dps gene, responsible for the glucan biosynthesis, was assayed by PCR. No amplification was achieved, indicative of the absence of these genes in the strains tested (data not shown).

Another concern is the production of BA, which are undesirable in all foods and beverages, as they can induce headaches, respiratory distress, hyper/hypotension and several allergic disorders (Ladero et al. 2010). BA are produced by some LAB by decarboxylation of precursor amino acids such as histidine, tyrosine, and ornithine, resulting in the formation of histamine, tyramine and putrescine, respectively, which are the most frequently BA found in wine (Lonvaud-Funel 2001; Ancín-Azpilicueta et al. 2008). PCR results demonstrated that the indigenous strains isolated in Caiño wine did not possess genes codifying for decarboxylase enzymes (hdc, tdc, odc genes). This result was also confirmed by TLC analysis, as production of BA by the studied bacteria in the presence of the BA precursor amino acids was not detected (data not shown).

Modification of wines after MLF

As displayed in Table 1, the initial total acidity was elevated in both wines. This acidity is characteristic of wines that are consumed young, as the ones studied here. After MLF, there was a moderate increase in pH values, but an important decrease in total acidity, which transformed the typical organoleptic characteristics of these wines.

Regarding colours parameters, it can be observed (Table 1) that in the case of Albariño wine the E420, S% and C* parameters had a light increase after MLF, meaning that the colour became more intense. The green component (a*) of the colour decreased, while the yellow one (b*) increased after MLF. This was observed also at a sensory level: the intensity of the yellow-golden highlights increased significantly (Fig. 5), while the transparency of the wine after MLF did not change (see Y% and L* values).

Figure 5.

Comparison of the sensory profile of Albariño before and after malolactic fermentation using common descriptors. a and b indicate significant differences (anova and Tukey's test, for P = 95%).

For Caiño wine, the colour was less intense than in the initial Albariño wine (at the end of the AF) and, after the sensory analysis, the colour was described by tasters as ‘straw yellow with yellow highlights’. After MLF there was, even in this case, an increase in the colour intensity (see E420, S% and C* values), but this was less evident for the panel (Fig. 6). There was also a decrease on the transparency of the wine (see Y% and L* values). On the other hand, phenolic composition of both wines was not affected by MLF.

Figure 6.

Comparison of the sensory profile of Caiño before and after malolactic fermentation using common descriptors. a and b indicate significant differences (anova and Tukey's test, for P = 95%).

The sensory profile of Albariño wine before MLF showed a straw yellow colour with yellow-golden highlights, all the odour descriptors – acacia flowers, citrus fruits, pineapple, pear, apple, aromatic herbs and honey – had a high intensity, the acidity was evident, the bitterness low, and it was considered a soft and high body product.

Figure 5 shows the evaluations of the common descriptors after comparison of the Albariño sensory profiles before and after MLF. The only significant difference was found for yellow-golden highlights, which increased after MLF as a consequence of the decrease in the acidity. Other differences, although not statistically significant, were observed for the odour descriptors: aromatic herbs and honey increased after MLF, while pineapple decreased. Two fruity odour descriptors, pear and apple, disappeared and the new descriptor vanilla were identified. A little improvement was reported for flavour descriptors, softness and body increased.

In the case of Caiño wine, before MLF the odour was characterized by high intensity of flowers (acacia flowers and orange blossom), tropical fruits, citrus fruits, banana, peach, pear, honey and aromatic herbs. As in the case of Albariño, in this product, the acidity was evident, the bitterness low, and it was considered a soft and high body product. It was shown that in Caiño, after MLF, ‘fresh’ notes such as tropical fruits, citrus fruits, banana, peach and pear disappeared, but a new odour descriptor caramel was identified. Statistical analysis performed on the common descriptors showed significant differences for acidity, aromatic herbs and honey (Fig. 6). Acidity decreased after MLF. Honey increased in wine after MLF, and this aspect gave to the wine more ‘mature’ notes, while aromatic herbs decreased. A not significant bitterness decreased was also observed after MLF.

Discussion

MLF is considered beneficial to the quality of the finished wine, and it is influenced by various factors. Principal among them is the presence and quantity of indigenous LAB that may include species from the genera Lactobacillus, Oenococcus, Pediococcus and Leuconostoc (Wibowo et al. 1985; Bae et al. 2006; Konig et al. 2009).

The selection of bacterial strains for the vinification process is principally based on its compatibility with the wine environment and the consumption of malic acid. In this regard, various works have been focused on the influence of different conditions, such as high ethanol concentration, low pH, or SO2 concentration on MLF and bacterial activity (Gockowiak and Henschke 2003; Alegria et al. 2004; Guzzon et al. 2009).

In white wines, MLF is not always carried out, because white wines are characterized by fresh notes and acid taste and, thus MLF, which reduces acidity, can be not desired for its effects. Consequently, Albariño and Caiño wines are usually bottled after AF without performing MLF. However, MLF could add beneficial flavour and, hence, starting from the same grapes, it could be possible to produce wines with different characteristics. For instances, an increase in the content of monoterpene, acetates and volatile phenols after MLF have been described in Albariño commercial wines from different origins (Falqué et al. 2008).

In the present report, the influence of MLF on sensory profile and organoleptic characteristics of Albariño and Caiño wines has been addressed for the first time. Microbiological characterization has shown that they contained bacteria belonging to Ped. damnosus species. Inoculation of these wines, which were not sterilized to simulate real winemaking conditions, with a commercial O. oeni starter to perform MLF showed that in Caiño wine MLF was accomplished by two indigenous Ped. damnosus strains that predominated over O. oeni starter, probably because the wine had a relatively high pH. By contrast, in Albariño neither the commercial O. oeni starter inoculated at the end of AF, nor the autochthonous population accomplished MLF, which was only completed after inoculation of Ped. damnosus strains isolated from Caiño wine. It is possible that the lack of spontaneous MLF in Albariño wine could be due to a low level of native bacteria at the end of AF. These data indicate that in these wines, which are characterized by high pH, the selected Ped. damnosus strains are particularly suitable starter for MLF.

This hypothesis was confirmed by subsequent tests conducted on sterilized Caiño wine, which showed that while MLF was not performed by Oenococcus, the selected autochthonous Ped. damnosus successfully do it, both alone and in the presence of O. oeni. Thus, these analyses conducted in sterilized wine that avoid the presence of active Pediococci demonstrated that the autochthonous Pediococci do not inhibit O. oeni growth, but that the specific wine characteristics are more probably responsible for the incapacity of O. oeni to complete MLF, even though this strain is a commercial starter widely used in the wineries. These results confirm that the selected Pediococcus strains are better adapted to this wine.

Pediococcus damnosus, Leuconostoc mesenteroides, and O. oeni have been identified as the key LAB accountable for MLF (Lonvaud-Funel 1999), but wine pH strongly influences which LAB species will be present. Higher pH wines (above pH 3·5) often harbor species of Lactobacillus and Pediococcus, both during and after fermentation, while lower pH wines (<3·5) typically contain only O. oeni (Fleet 1998; Osborne and Edwards 2005). In fact, the frequent occurrence of Pediococcus spp. in Australian wines has been proposed to be indicative of a high concentration of SO2 and a high pH (Costello et al. 1983), as Pedcerevisiae (now Ped. damnosus) was dominant in the wines analysed (45 reds and 24 whites) and O. oeni population disappeared. Likewise, Davis et al. (1986) also showed that the pH had a profound, selective effect upon the species that grow in wines, as O. oeni was usually the only one isolated from wines with a pH below 3·5, in which Pediococcus and Lactobacillus spp. rarely grow, although they do it in wines with pH over 3·5 (Ribereau-Gayon et al. 1975; Davis et al. 1986). Thus, growth of these bacteria seems to be antagonistic to O. oeni survival (Wibowo et al. 1985; Davis et al. 1986). Chemical and biomolecular assessment of the possible negative characteristics usually attributed to Ped. damnosus species of the autochthonous strains isolated here from Caiño wine demonstrated that they did not produce BA or exopolysaccharides.

Once the lack of the above-mentioned negative characteristics of the selected Pediococcus strains was verified, the wines were evaluated with sensory analysis by a trained panel of tasters before and after MLF. This analysis indicates that the MLF induced in Albariño both an aromatic change and a significant difference in the colour, which became more yellow-golden, as well as a decrease in the acidity. Moreover, after MLF, there was an increase in the body and softness of this wine. On the other hand, Caiño wine that underwent MLF was described as more mature than the original one showing a lower acidity.

The presence of volatile compounds and sensory analyses of Albariño musts and wines have been already reported (Oliveira et al. 2008; Vilanova et al. 2008, 2010), but, until now, no previous studies on the effect of MLF on this wine or on the sensory profile of Caiño wine has never been made. The analysis of these profiles showed that MLF gave new characteristics to the wines without affecting flower descriptors. Therefore, and even though, to maintain their fresh notes, MLF is not usually allowed to occur in white wines, on the bases of the results present in this study, it can be useful to wine industry for the production of wines having more complex characteristics.

In the last two decades, and albeit the use of malolactic starter cultures has become widespread to carry on the MLF, the homofermentative species Ped. damnosus has not being used because it is considered a spoilage micro-organism. However, our results indicate that the strains isolated in this report are useful starters for the MLF in wines that, as Albariño and Caiño, are characterized by a high pH value. Supporting this, it has been reported that MLF significantly contribute to the formation of volatile aroma compounds in white wines, as Chardonnay, and that pH and ethanol content were important factors influencing the volatile aroma composition (Knoll et al. 2011). Even more, the same study also suggest that the choice of the starter is very important to obtain a wine with the desired final characteristics, as it can influence the final chemical composition and the sensory profile of the wine. Likewise, several strains of Pediococcus spp were isolated from wines that were not considered spoiled, but on the contrary they were judged to be of good quality (Edwards and Jensen 1992).

As commented previously, there is an increased interest to investigate new species for MLF. In this respect, and although Pediococcus is usually considered a spoilage microorganism together with Lactobacillus species, some researchers have isolated Pediococcus from wines that were not considered spoiled. For example, Pediococcus parvulus altered the bouquet of a non-MLF Cabernet Sauvignon, but did not spoil it (Osborne and Edwards 2005), thus confirming that some pediococci may add desirable flavours and aromas to the wine. In agreement with that, our results indicate that in wines with high pH, as Albariño and Caiño, Pediococcus is well adapted to perform MLF. Even more, under a practical point of view, an important characteristic of Pediococcus is its higher growth rate, making its employment an interesting option in winemaking.

In conclusion, this study demonstrated that MLF can positively affect Albariño and Caiño wines, and that the bacterial indigenous population of these wines was composed by Pediococcus, which can successfully perform MLF without negative effects on the wine, as they did not produce BA or exopolysaccharides. Therefore, Ped. damnosus strains can be considered as a new topic of investigation on malolactic starter.

Acknowledgements

This study was funded through Projects Bodega Terras Gauda LTD. Xunta de Galicia (PGIDIT04TAL035E), 2004-7-OE-242, AGL2006-02558, A36108900, ALIBIRD-CM-S-0505/AGR-0153, and CONSOLIDER INGENIO 2010 (CSD2007-00063FUN-C-FOOD). The study was also partially supported by BIODATI project DM 16101/7301/08. We would like to thank Emilio Rodriguez Canas and Terras Gauda S.A for their assistance in the experimental work. We are grateful to Dr. J.C. Saiz for the critical reading of the manuscript.

Conflicts of Interest

No conflict of interest declared.

Ancillary