Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation.

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Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation Pedro Miguel Izquierdo Cañas, Esteban García Romero, Fátima Pérez-Martín, Susana Seseña and María Llanos Palop Food Science and Technology International published online 28 February 2014 DOI: 10.1177/1082013214524585 The online version of this article can be found at: http://fst.sagepub.com/content/early/2014/02/28/1082013214524585

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Research Article

Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation ˜ as1,2, Esteban Garcıá Romero1, Pedro Miguel Izquierdo Can ´ tima Pe ´ rez-Martıń3, Susana Sesen ˜ a3 and Marıá Llanos Palop3 Fa

Abstract A study has been carried out in order to determine the effect of the lactic acid bacteria inoculation time on the kinetic of vinification and on chemical and sensory characteristics of Cabernet Franc wines. Traditional vinifications, with lactic acid bacteria inoculated after completion of alcoholic fermentation were compared with vinifications where yeast and bacteria were co-inoculated at the beginning of vinification. One commercial yeast strain and an autochthonous Oenococcus oeni strain (C22L9), previously identified and selected at our laboratory, were used. Monitoring of alcoholic and malolactic fermentations was carried out by yeast and lactic acid bacteria counts and by measuring L-malic acid concentration. Wines were chemically characterized and analysed for volatile compounds content. A sensory analysis, consisting of a descriptive and a triangular test, was also carried out. Results from this study showed that the concurrent yeast/bacteria inoculation of musts at the beginning of vinification produced a reduction in duration of the process without an excessive increase in volatile acidity. Differences in volatile compounds content and the corresponding impact on the sensorial profile of wines were also displayed. These results suggest that co-inoculation is a worthwhile alternative for winemaking of Cabernet Franc wines, if compared with traditional post-alcoholic fermentation lactic acid bacteria inoculation.

Keywords Malolactic fermentation, co-inoculation, sequential inoculation, volatile compounds, red wine Date received: 20 September 2013; accepted: 17 January 2014

INTRODUCTION Winemaking is a complex process frequently involving two successive fermentations: an alcoholic fermentation (AF), conducted by yeasts, and a subsequent malolactic fermentation (MLF) carried out by wine lactic acid bacteria (LAB). MLF results in deacidification and microbial stabilization of wine, and in addition it produces changes at the organoleptic profile of wines which have important consequences for the final quality (Bauer and Dicks, 2004; Lonvaud-Funel, 1999). Both AF and MLF may occur spontaneously from the activity of yeasts and bacteria naturally present in Food Science and Technology International 0(0) 1–10 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013214524585 fst.sagepub.com

musts and wines, although in the last years winemakers are starting to recognize the benefits of inoculating with dried commercial starter cultures of yeast and LAB for better control of how and when these fermentations take place. Timing of inoculation of starter cultures is an important factor influencing the success of induced 1

Instituto de la Vid y el Vino de Castilla-La Mancha, Tomelloso (Ciudad Real), Spain 2 ´ gico de Albacete, Albacete, Spain Parque Cientı´fico y Tecnolo 3 Departamento de Quı´mica Analı´tica y Tecnologıá de Alimentos, Facultad de Ciencias Ambientales y Bioquı´mica, Universidad de Castilla-La Mancha, Toledo, Spain Corresponding author: Susana Sesen ˜ a, Departamento de Quı´mica Analı´tica y Tecnologıá de Alimentos, Facultad de Ciencias Ambientales y Bioquı´mica, Universidad de Castilla-La Mancha, Avda. Carlos III s/n, 45071 Toledo. Email: [email protected]



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Food Science and Technology International 0(0) fermentations, and various studies have been carried out to determine the effect of bacterial inoculation time on vinification kinetics, chemical composition and sensory and sanitary attributes of wines (Abrahamse and Bartowsky, 2012; Alexandre et al., 2004; Antalick, 2010; Izquierdo Canãs et al., 2012). Results from some of these studies have shown that simultaneous yeast/bacteria inoculation has important risks, such as the development of undesirable/antagonistic interactions between the two microorganisms, stuck AF, interruption of AF before sugar depletion, wines with increased concentrations of acetic acid that render them unacceptable for consumption or the production of possible off-odours (Mendoza et al., 2011). Consequently, simultaneous inoculation has not been a very common practice and the addition of bacterial starter culture after the completion of AF has been largely adopted by wineries. On the contrary, other authors (Izquierdo Canãs et al., 2012; Krieger et al., 2007; Sieczkowski, 2004) have recommended simultaneous addition of yeasts and bacteria to the must, on the basis of a better performance of the bacteria, due to the low alcohol concentration, the higher nutrient availability present in musts and to better sensory characteristics of the wines. Likewise, others (Azzolini et al., 2010; Massera et al., 2009; Zapparoli et al., 2009) have reported a reduction in total fermentation time and better control of the MLF due to the early dominance of the inoculated bacterial strain. However, coinoculation is not yet a very common practice and new and more exhaustive studies are needed to gain more knowledge of the influence of early bacterial inoculation on the winemaking process and on wine quality. The aim of this study is to know the influence of LAB inoculation time on vinification kinetics and on the chemical composition and sensory characteristics of Cabernet Franc wines. In this purpose, traditional vinifications, with LAB inoculated after completion of AF, were compared with vinifications where yeast and bacteria were inoculated concurrently. To our knowledge, works reporting such comprehensive survey on the effect of timing of inoculation of the LAB in the vinification process of this red wine variety have not been published to date.

MATERIALS AND METHODS Microorganisms Saccharomyces cerevisiae strain Uvaferm VNÕ and the Oenococcus oeni strain C22L9, an autochthonous strain isolated and selected at our lab from Tempranillo wine (Ruiz et al., 2010) were obtained as dried culture from Lallemand (Montreal, Canada).

Fermentation assays Cabernet Franc grapes from La Mancha wine region (Spain) were harvested during the 2011 vintage. Musts were obtained in the experimental cellar of the Institute of Vine and Wine of Castilla-La Mancha. Chemical composition of must was determined following the official analytical methods established by the International Organisation of Vine and Wine (OIV, 2011) as follows: 13.92 Baume´; 4.31 g/l total acidity; 3.17 pH and 1.60 g/l L-malic acid. A total of 50 mg/l of SO2 were added to must for sequential inoculation assays while 40 mg/l SO2 were added to that for coinoculation assays. This concentration is compatible with bacteria growth, provided that the bacteria are inoculated some hours after SO2 addition to allow the combination of the SO2 (Ribe´reau-Gayon et al., 2006). Each fermentation type (co-inoculation and sequential) was carried out in triplicate using 100 kg of grapes each and following the standard protocols for Cabernet Franc vinifications. The yeast, S. cerevisiae VNÕ , and the malolactic bacteria, O. oeni C22L9, were inoculated according to the manufacturer’s instructions. LAB was inoculated either after the completion of AF, when Glucose þ Fructose (G þ F) content was below 1 g/l (sequential), or 24 h after yeast inoculation (co-inoculation), when free SO2 concentration was less than 10 mg/l. At the end of MLF wines were decanted and sulphited to reach 25 mg/l of free SO2 and then clarified, stabilized and filtered through 0.2 mm filters before bottling. Microbiological analysis Serial 10-fold dilutions of samples taken after inoculation and until the end of MLF were spread on Malt Extract Agar (Cultimed, Barcelona, Spain) for yeast counts and on MLO Agar (MLOA, Oenococcus oenos Medium) (Scharlab, Barcelona, Spain) supplemented with 10% (v/v) tomato juice, 50 mg/ml sodium azide and 100 mg/ml cycloheximide, for LAB counts. Malt extract agar plates were incubated at 28 C for 48 h and MLO plates were incubated at 30 C for 5 days under anaerobic conditions (Gas Pack System, Oxoid Ltd, Basingstoke, UK). Counts were performed in duplicate and expressed as colony forming units (CFU) per ml of wine. An implantation study, as described by Izquierdo Canãs et al. (2012), was performed to control the presence of the inoculated O. oeni C22L9 during fermentations. Chemical analysis Wines were chemically characterized by determining alcohol content; total acidity (expressed as tartaric

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Izquierdo Can ãs et al. acid); pH; volatile acidity (expressed as acetic acid); Lmalic acid, L-lactic acid and citric acid contents; total SO2; colour intensity (CI) and tonality. The official analytical methods established by the International Organisation of Vine and Wine (OIV, 2011) were used for this characterization. MLF was monitored following L-malic acid degradation. Anthocyanins were determined by decolouring with sulphuric acid (Ribe´reau-Gayon and Stronestreet, 1965) and total polyphenols by measurement of absorbance at 280 nm following conventional dilution of the sample (Somers and Evans, 1977). Total flavan-3-ols (procyanidins) were determined by reaction with dimethylaminocinnamaldehyde and measurement at 640 nm (Nagel and Glories, 1991; Vivas et al., 1994), and the tannins by precipitation with methylcellulose (Smith, 2005). Volatile compound analysis Volatile compounds were analysed by gas chromatography–mass spectrometry (GC–MS) using a ThermoQuestGC2000 gas chromatograph and a DSQII mass detector with quadrupole analyser. All masses were obtained in electronic impact mode at 70 eV. A BP21 column (SGE) 50 m 0.32 mm internal diameter and 0.25 mm thick of Free Fatty Acid Phase (FFAP) (polyethylene glycol treated with nitroterephthalic acid) was used. For the major volatile compounds, 200 ml of wine was steam distilled as described by OIV (2011). One microlitre of distilled wine with 4-methyl-2-pentanol (final concentration 20 mg/l) as internal standard was directly injected. The chromatographic conditions were as follows: carrier helium gas (1.7 ml/min, split 1/25); injector temperature, 220 C and oven temperature, 43 C for 5 minutes, 4 C/min to 100 C, 20 C/ min to 190 C, and 45 min at 190 C. Minor volatile compounds were extracted using the method developed by Ibarz et al. (2006). Twenty five millilitres of wine were passed through a preconditioned polypropylene–divinylbenzene cartridges (0.2 g of Lichrolut EN (40–120 mm), Merck) using 4-nonanol as internal standard (final concentration 250 mg/l). The column was rinsed with 25 ml of water to eliminate sugars, acids and other polar compounds. The free fraction of volatile compounds was eluted with 15 ml of pentane–dichloromethane (2 : 1 v/v). Extracts were concentrated by distillation in a Vigreux column and under nitrogen stream to 100 ml and then kept at 20 C until analysis. Separated compounds were identified by their mass spectra and their chromatographic retention times, using commercial products as a standard. Quantification was performed by analysing the

characteristic m/z fragment for each compound using the internal standard method. Results for non-available products were shown as the relationship between the area of each compound and that of the internal standard. Sensory analysis Sensory analysis was performed to investigate the differences among the two different inoculation procedures. Wine samples from triplicates of both fermentation types were evaluated using both triangular and descriptive tests. A triangular test to evaluate the differences in aroma and taste was conducted in dark wine-tasting glasses to avoid judgements being influenced by the colour of the wine. Sets containing three samples were analysed by 14 panelists in three sessions carried out at different days, according to ISO Standard 4120 (ISO, 1983). Descriptive sensory analysis was performed by 10 selected panelists following the Sensory Profile method according to ISO Standard 11035 (ISO, 1994). The descriptors were scored on a scale of 0–5 (0 absence of the descriptor and 5 maximum intensity of the descriptor). The odour attributes evaluated were aromatic intensity, ripe fruit, fresh fruit, floral, dairy, vegetable, aromatic plants, spicy and balsamic. In addition, the gustative phase attributes were aftertaste intensity, body and bitter taste. From results of this analysis, a consensus profile was carried out by the panelists. Statistical analysis The paired Student’s t-test was used to determine whether there were significant differences between the results from chemical and volatile compound analysis. Principal Component Analysis (PCA) was used to obtain a more comprehensible overview of the chemical compounds and to investigate possible correlations amongst wines. The SPSS 12.0 software was used for both analyses.

RESULTS AND DISCUSSION Evolution of L-malic acid and microbial populations Figures 1 and 2 show yeast and LAB counts and evolution of L-malic acid from sequential and co-inoculation assays, respectively. Initial microbial counts at musts were identical at both assays in spite of the different SO2 concentrations added. Viable yeast population followed a similar evolution at sequential and coinoculation assays, until beginning of MLF. As reported by other authors (Massera et al., 2009; 3




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Food Science and Technology International 0(0)

1,0E+09

2,00

1,0E+08

1,0E+06 cfu/mL

1,20 1,0E+05 1,0E+04 0,80 1,0E+03 1,0E+02

L-malic acid (g/L)

1,60

1,0E+07

0,40

1,0E+01 0,00

1,0E+00 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Time (days) Yeasts counts

Lactic acid bacteria counts

L-malic acid

Figure 1. Yeasts and LAB counts and evolution of L-malic acid from sequential inoculation assays. Arrow indicates bacterial inoculation in sequential treatment. Values are mean of triplicates SD.

1,0E+09

2,00

1,0E+08 1,60

cfu/mL

1,0E+06

1,20

1,0E+05 0,80

1,0E+04

L-malic acid (g/L)

1,0E+07

1,0E+03 0,40 1,0E+02 1,0E+01

0

2

4

6

Yeasts counts

8

10

12

14

16

18

20

22

Time (days) Lactic acid bacteria counts

24

26

28

0,00

L-malic acid

Figure 2. Yeasts and LAB counts and evolution of L-malic acid from co-inoculation assays. Arrow indicates bacterial inoculation in co-inoculation treatment. Values are mean of triplicates SD.



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Izquierdo Can ãs et al. Table 1. Chemical composition of Cabernet wines at the end of MLF

Alcohol content (% v/v) Total acidity (g/l) pH Volatile acidity (g/l) L-malic acid (g/l) L-lactic acid (g/l) Citric acid (g/l) Total SO2 Colour Intensity Tonality Free anthocyanins (mg/l malvidin) Polymerized anthocyanins (mg/l malvidin) Total polyphenols (mg/l gallic acid) Catechins (mg/l catechins) Tannins (g/l)

Sequential

Co-inoculation

14.32 0.11 4.22 0.05 3.47 0.02 0.49 0.03 0.05 0.01 0.97 0.04 0.19 0.01 59.00 13.08 11.16 0.31 0.662 0.002 360.07 3.68 54.23 2.61 1251.47 25.41 144.97 7.88 1.40 0.13

14.40 0.39 4.22 0.07 3.44 0.04 0.49 0.02 0.07 0.02 1.06 0.03* 0.26 0.03* 50.33 4.04 10.81 0.49 0.631 0.014* 403.13 10.01* 40.43 2.78* 1221.80 36.76 146.00 4.24 1.35 0.29

Values are the mean of triplicates. *Statistically significant differences (p 0.05) between the different inoculation methods.

Mendoza et al., 2011), the presence of O. oeni during active AF in co-inoculation assays seems not to influence the viable yeast population, since the counts obtained from both time inoculation assays were similar. As expected, evolution of LAB population was different for both assays. In co-inoculation assays, counts around 105 CFU/ml were obtained immediately after inoculation and up to day 10, when a gradual increase was observed until the end of the vinification process. In concordance with the increase of LAB counts observed at day 10, a decrease in the L-malic acid content was observed from this day to the end of the process. In sequential inoculation assays, counts of LAB during AF were much lower and only until the inoculation of O. oeni, at day 16 of the vinification process, values were similar to those of co-inoculation assays. At the end of MLF, LAB counts reached values around 107 CFU/ml at both assays. Differences in overall fermentation (AF þ MLF) duration were observed between sequential and coinoculation assays, with values ranging between 32 and 28 days, respectively (Figures 1 and 2). As reported by Izquierdo Canãs et al. (2012) for Tempranillo and Merlot wines, the time required for sugar concentration to fall below 1 g/l (data not shown) and L-malic acid concentration below 0.1 g/l (Figures 1 and 2) was shorter in co-inoculation assays, though differences between both inoculation times have not been so important in this study. However, the shorter time required to conclude the vinification process when

using co-inoculation has importance from a technological point of view. Also as reported by Izquierdo Canãs et al. (2012), Lmalic acid degradation did not start until LAB population reached values around 106 CFU/ml. Implantation of O. oeni C22L9 was 100% in all samples taken after LAB inoculation. Chemical analysis of wines Table 1 summarizes the mean values standard deviation of the chemical parameters analysed in the wines from the triplicate vinifications. Values for citric acid concentrations in wines from co-inoculation assays were significantly higher than those from sequential inoculation. Likewise, concentration of L-lactic acid was significantly higher in wines from co-inoculation assay. On the contrary, values for volatile acidity, expressed as acetic acid, were identical at wines from both assays. This value is considered to conform to the standard quality parameter for volatile acidity in red table wine (Azzolini et al., 2010; Ribe´reau-Gayon et al., 1989). A slight but undesirable decline in the concentration of free anthocyanins in the wine during MFL has been reported by some authors (Ruiz et al., 2012). Results from this study show that the co-inoculation process keeps anthocyanin concentration in a statistically higher value than the sequential inoculation while polymerized anthocyanin concentration was statistically lower. 5



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Food Science and Technology International 0(0) Volatile compound analysis Table 2 presents the mean and the standard deviation values for the volatile compounds determined by GC–MS. With respect to lineal alcohols, C6 alcohols and bencenic alcohols, it is important to highlight that wines produced by co-inoculation contained significantly higher propanol concentration and significantly lower concentrations of 1-octen-3-ol, 2-phenylethanol and tyrosol, compounds which contribute significantly to wine aroma (Ugliano and Henschke, 2008). Esters are also important compounds influencing wine aroma, and the presence of some short-chain esters, such as ethyl acetate, isobutyl acetate, isoamyl acetate and hexyl acetate, contributes imparting fruity flavours. Others such as diethyl succinate and ethyl lactate impart fruity, buttery and creamy notes and contribute to mouthfeel (Izquierdo et al., 2008; Lerm et al., 2010). Ester synthesis and hydrolysis during MLF are due to the esterase activity of lactic acid bacteria and there is disagreement among authors about the influence of MLF on the final ester content (Boido et al., 2009). Differences in the behaviour of esters were observed for wines from both assays. So, wines produced by co-inoculation contained higher concentrations of ethyl acetate, hexyl acetate, ethyl lactate, ethyl butyrate, ethyl hexanoate, ethyl octanoate and diethyl succinate. Concentrations of the most of these compounds differed significantly from those for the same compounds in sequential wines. On the contrary, concentrations of 2-phenylethyl acetate, ethyl dodecanoate and diethyl malate were significantly lower in wines from co-inoculation assays. Concentration of acetaldehyde was also higher in co-inoculation wines while similar concentrations of 2,3-butanodione and 3-hydroxy-2-butanone were obtained for wines from both assays. Moderate concentrations of these compounds give the wine aromatic complexity, with buttery notes, contributing positively to aroma and organoleptic quality (Izquierdo Canãs et al., 2012). Of the group of volatile phenols, ethyl phenols are particularly important because they contribute negatively to the final quality of wine, being responsible for the ‘phenolic’, ‘animal’ and ‘stable’ off-odours found in certain red wines (Gerbaux et al., 2009). Results from this study displayed significantly lower concentrations of 4-vinyl-guaiacol and 4-vinylphenol (precursors of ethyl phenols) in wines produced by co-inoculation, in concordance with Izquierdo Canãs et al. (2012). On the contrary, ethyl phenol concentration was higher in co-inoculation wines although differences were not significant.

Terpenes are compounds present in free and glycosylated form in grapes. During AF, content of free terpenes often increases due to the b-glucosidase activity of yeasts (Gil et al., 1996) and during MLF a decrease of glycosidically bound volatile compounds, terpenes included, occurs (Ugliano and Moio, 2006). The ability of O. oeni strains to release terpenes from glycosidic precursors has been described by some authors (D’Incecco et al., 2004; Hernańdez-Orte et al., 2009) with the degree to which the enzymatic hydrolysis takes place being dependent on the bacterial strain, the chemical structure of the substrate and the growth phase of the bacteria (Lerm et al., 2010; Ugliano and Moio, 2006). For these compounds only slight differences in the hydroxycitronellol and geraniol concentrations were observed between wines. Norisoprenoids also exert a significant influence on the sensory quality of wines (Izquierdo et al., 2008) contributing with fruity, floral or spicy notes. Concentrations of some of these compounds, such as 3-oxo-a-ionol, dihydro-a-ionone and 3-oxo-7,8-dihydro-a-ionol were statistically lower in the co-inoculation wines. The last group of compounds analysed correspond to the methoxyphenols, which give wines spicy and smoked characteristics (Ferreira et al., 1995). It is worth noting the results for vanillin, zingerone, homovanillyl alcohol and siringol, concentrations of which were significantly lower in the wines produced by coinoculation. Multivariate data analysis Principal component analysis (PCA) was applied to the results from chemical and volatile compound analysis. Figure 3 shows the variables best correlated with principal component 1 (PC1) and principal component 2 (PC2) and the distribution of wines on the plane formed by these two principal components. A 76.003% of the variance was explained by the first two principal components. Two different groups were evident: the co-inoculation wines on the negative side of the PC1 and PC2 and the sequential wines located on the positive side of these axes. Co-inoculation wines had higher concentrations of some important esters (isoamyl acetate, ethyl butyrate, ethyl hexanoate, diethyl succinate, ethyl acetate) and acetaldehyde while sequential wines had higher concentrations of 2phenylethanol, 2-phenylethyl acetate, 4-vinyl-guaiacol and 4-vinylphenol. Sensory analysis Results from the triangular test showed that the panelists detected statistically significant differences between wines, at 95% of probability. Total number of correct



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Izquierdo Can ãs et al. Table 2. Volatile compound concentrations in Cabernet Franc wines at the end of MLF Sequential

Co-inoculation

Lineal alcohols 48.7 4.02

a

42.8 2.52

Butyric acidb

20.7 1,17

23.7 0.68*

Isobutyric acid

46.3 1.88

45.7 1.13

Valeric acidb

Active amyl and isoamyla

285.8 9.18

274.4 4.66

1-Pentanolb

101.4 5.72

Isobutanola

L-2,3-butanodiol

Co-inoculation

509.1 61.4

459.3 75.9

867.3 102.6

820.9 50.8

Acids

Methanola Propanol

Sequential

b

96.2 10.72

b

39.2 1.14

45.9 2.96*

Isovaleric acidb

109.0 5.32

102.9 3.56

Hexanoic acida

2.19 0.24

2.17 0.41

a

9.29 5.52

3.35 0.85

Octanoic acid

2.76 0.29

2.56 0.58

Meso-2,3-butanodiolb

25.3 7.01

17.9 4.37

Decanoic acida

1.56 0.16

1.32 0.83

3-Ethoxy-1-propanolb

57.9 3.28

70.4 7.34

Phenylacetic acidc

20.5 5.63

7.92 3.97*

1-Octen-3-olb

4.21 0.15

3.76 0.22*

Furanic compounds

6-Methyl-5-hepten-2-olb

4.08 0.32

3.80 0.40

Furfuryl alcoholb

3.56 0.99

3.45 0.58

Furfuralb

6.69 0.36

4.13 0.15*

0.94 0.07

0.81 0.05

102.1 9.11

51.1 12.5*

Phenolb

4.18 0.13

3.65 0.36

4-Ethyl-phenolb

0.45 0.14

0.80 0.47

4-Ethyl-guaiacolb

0.25 0.08

0.20 0.07

205.4 28.25

93.01 30.11*

7.58 0.36

3.86 0.75*

Linalolb

3.64 0.41

3.16 0.05

Hydroxycitronellolc

6.99 1.07

5.87 0.58

C6 Alcohols 1-Hexanola

4.75 0.43

4.60 0.04

2-Acetyl-furanec

c-2-Hexenolb

9.47 0.57

9.60 0.28

Hydroxymethylfurfuralb

273.7 16.1

280.8 18.9

14.1 1.83

16.1 3.44

114.4 15.81

110.1 1.55

c-3-Hexen-1-olb t-2-Hexenolb t-3-Hexenol

b

Bencenic alcohols Benzyl alcohola 2-Phenylethanola Tyrosol

a

0.27 0.02

0.27 0.01

4-Vinyl-guaiacolb

103.0 4.68

84.9 5.49*

4-Vinylphenolb

7.08 0.67

3.73 0.71*

Terpenes

Acetates Ethyl acetatea

17.9 2.29

Isoamyl acetate

a

Volatile phenols

24.6 3.33*

b

0.48 0.09

0.73 0.11

Citronellol

9.93 0.47

9.67 1.69

101.8 2.45

91.6 4.81*

Geraniolb

10.4 0.11

11.3 1.76

Butyl acetateb

2.83 1.04

2.07 0.71

3,7-Dimetil-1,5-octadien-3, 7-diolc

8.93 1.39

8.12 2.14

Hexyl acetateb

0.78 0.06

1.05 0.11*

Norisoprenoids

Benzyl acetateb

0.45 0.05

0.43 0.08

Damascenoneb

5.88 0.76

5.29 0.70

0.44 0.06

0.34 0.08

2-Phenylethyl acetateb

Ethyl esters

b-ionone

Ethyl lactatea

13.1 0.86

17.1 0.44*

Ethyl butyrateb

82.4 1.50

123.2 16.4*

Ethyl isovaleratec

0.13 0.01

0.14 0.06

a

b

3-OH-b-damasconec 3-Oxo-a-ionolc Dihydro-a-iononec c

1.93 0.33

1.41 0.27

172.6 17.2

128.7 13.9*

4.61 0.36

3.04 0.51*

0.24 0.08

0.31 0.03*

3-OH-7,8-dihydro-b-ionol

2.07 0.35

1.63 0.06

Ethyl octanoatea

0.25 0.06

0.28 0.01*

3-Oxo-7,8-dihydro-a-ionolc

8.79 1.84

4.43 1.32*

Ethyl decanoateb

44.9 1.90

47.1 1.45

b-ionolb

4.13 0.16

3,59 1.12

Ethyl dodecanoateb

1.34 0.25

0.81 0.08*

Methoxiphenols

Ethyl hexanoate

Guaiacolb

Other esters 2-Ethyl furoate

b

2.20 0.21

2.06 0.20

2.10 0.08

1.76 0.27

b

34.2 6.58

18.9 3.39*

Ethyl vanillatec

1.56 0.61

3.75 2.04

6.65 1.15

4.85 0.77

Vanillin

2-Phenylethyl-succinatec

10.5 1.63

5.9 1.22*

Diethyl malatec

72.1 9.27

35.8 8.99*

Methyl vanillateb

Ethyl monosuccinatec

20.4 1.74

7.64 5.68

Zingeroneb

11.70 1.63

3.36 1.43*

Isoamyl octanoatec

0.47 0.06

0.53 0.08

Homovanillyl alcoholb

122.7 4.33

68.19 23.7*

Methyl octanoate

0.25 0.02

0.26 0.03

Acetovanilloneb

56.2 6.87

46.3 2.53

Methyl salicilateb

2.44 0.32

2.49 0.17

Propiovanilloneb

5.73 1.33

5.11 0.24

Diethyl succinatea

0.46 0.04

0.60 0.06*

Acetosyringoneb

23.7 6.80

12.0 1.08

b

(continued)



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Food Science and Technology International 0(0) Table 2. Continued Sequential

Co-inoculation

Carbonilic compounds Acetaldehydea 2,3-Butanodione

Eugenol 4.64 0.20

a

Sequential

5.28 0.31*

4.31 0.19

4.47 0.29

3-Hydroxy-2-butanonea

1.09 0.25

1.50 0.30

2-Methyl-tetrahydrothiophene-3-onec

12.6 1.56

23.1 3.74*

b

Siringolb 2-Methoxy-benxyl alcohol

c

Co-inoculation

3.29 0.35

2.83 0.49

15.8 2.10

11.2 1.78*

1.76 0.16

1.37 0.48

Values are the mean of triplicates. a mg/l. b mg/l. c Area compound/area internal standard. *Statistically significant differences (p 0.05) between the different inoculation method.

Figure 3. Plotting of the samples and the variables best correlated on the plane defined by the two principal components obtained by principal component analysis (PCA) of the data from chemical and volatile compounds.

responses for aroma and taste was 28 and 31, respectively (total number of responses ¼42). These differences may be attributable to the longer aftertaste intensity and body in sequential wines and to the more aromatic intensity and vegetable aroma of co-inoculation wines. Then a descriptive sensory analysis of wines, in order to know attributes responsible of those differences, was performed. As can be observed in Figure 4 sensory profiles of wines were similar with only slight differences attributable to the type of inoculation. It seems to be that wines produced by co-inoculation had more aromatic intensity, floral and vegetable aroma while

sequential wines had more intense spicy aroma and a longer aftertaste intensity and body.

CONCLUSIONS The current study has shown that co-inoculation of the commercial yeast strain Uvaferm VNÕ and the lactic acid bacteria strain O. oeni C22L9 is a real alternative to the traditional winemaking for Cabernet Franc wines. It represents a saving of time for wineries (4 days) and no evidence of a negative impact on fermentation success or on final wine parameters has been



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Izquierdo Can ãs et al.

Aromatic intensity 5 4.5 4

Bitter

Ripe fruit

3.5 3 2.5

Body

Fresh fruit

2 1.5 1 0.5 Floral

0

Aftertaste intensity

Balsamic

Dairy

Spicy

Vegetable Aromatic plants Sequential

Coinoculation

Figure 4. Descriptive sensory analysis.

found. These are important advantages for wineries, in terms of process efficiency, and, in addition, the presence of a selected LAB strain from the outset of microvinification keeps out other undesirable spontaneous bacteria, thus conferring sanitary benefits. Our results also have confirmed the findings of other authors (Azzolini et al., 2010; Izquierdo Canãs et al., 2012) for different grape varieties, demonstrating the possibility of simultaneous induction of alcoholic and MLF without an excessive increase in volatile acidity. Exhaustive chemical analysis revealed numerous differences as regards volatile compound composition, and multivariate analysis of these results clearly separated wines according to the inoculation mode. These differences were also observed on the sensory profiles of wines, and panelists detected higher aromatic intensity and vegetable aroma at wines obtained from coinoculation. FUNDING This work was financed by Consejerı´ a de Educacioń y Ciencia of the Junta de Comunidades de Castilla-La Mancha (JCCM) for project PCC 05-003-2 and Ministerio de Educacioń y Ciencia (INIA) for project RM 2006-00011-C02-02.

ACKNOWLEDGEMENTS F. Pe´rez-Martı´ n is supported by a grant of the Council of Communities of Castilla-La Mancha cofounding by

European Social Fund. P. M. Izquierdo acknowledges the European Social Fund and INCRECYT for cofunding his contract. We also thank J. M. Heras at Dantars Ferment A. G. for donation of the Oenococcus oeni and yeast strains.

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