Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts

Accepted Manuscript Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts Carlos Escott, Juan Manuel Del Fresno, Iris Loira, Antonio Morata, Wendu Tesfaye, María del Carmen González, José Antonio Suarez-Lepe PII: DOI: Reference:

S0308-8146(17)31181-0 http://dx.doi.org/10.1016/j.foodchem.2017.07.037 FOCH 21421

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

4 April 2017 30 June 2017 10 July 2017

Please cite this article as: Escott, C., Fresno, J.M.D., Loira, I., Morata, A., Tesfaye, W., González, M.d.C., SuarezLepe, J.A., Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem. 2017.07.037

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Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts

Carlos ESCOTT*, Juan Manuel DEL FRESNO, Iris LOIRA, Antonio MORATA, Wendu TESFAYE, María del Carmen GONZÁLEZ, José Antonio SUAREZ-LEPE

enotecUPM. Chemistry and Food Technology Department. School of Agronomic, Food and Biosystems Engineering. Technical University of Madrid. Av. Puerta de Hierro 2, 28040 Madrid, Spain.

*Corresponding author. Tel.: +34 91 336 57 30; Fax: +34 91 336 57 46 E-mail address: [email protected] Abstract Non-Saccharomyces yeasts may contribute to enrich wine aroma while promoting the formation of stable pigments. Yeast metabolites such as acetaldehyde and pyruvate participate in the formation of stable pigments during fermentation and wine aging. This work evaluated the formation of polymeric pigments in red musts added with (+)Catechin, ProcyanidinB2 and ProcyanidinC1. The non-Saccharomyces yeasts used were Lachancea thermotolerans, Metschnikowia pulcherrima and Torulaspora delbrueckii in sequential fermentation with Saccharomyces cerevisiae and Schizosaccharomyces pombe. Use of Lachancea thermotolerans led to larger amounts of polymeric pigments in sequential fermentation. (+)-Catechin is the flavanol prone to forming such pigments.

1

The species Metschnikowia pulcherrima produced higher concentration of esters and total volatile compounds. The sensory analysis pointed out differences in fruitiness and aroma quality. The results obtained strengthen the fact that metabolites from nonSaccharomyces yeasts may contribute to form stable polymeric pigments while also influencing wine complexity.

Keywords:

procyanidins,

polymeric

pigments,

pyranoanthocyanins,

non-

Saccharomyces, red wine. Chemical compounds studied in this article: (+)-Catechin (PubChem CID: 73160); Procyanidin B2 (PubChem CID: 122738); Procyanidin C1 (PubChem CID: 169853); Pyruvic acid (PubChem CID: 1060); Acetaldehyde (PubChem CID: 177); Malvidin-3O-glucoside (PubChem CID: 443652); Vitisin A (PubChem CID: 16131430); Vitisin B (PubChem CID: 16138152); Malvidin glucoside-ethyl-catechin (PubChem CID: 71308233).

2

1. Introduction Regarding red wines colour, phenomena like the copigmentation of anthocyanins may contribute to more than 30 % of the colour for fresh red wines (Bimpilas et al. 2016) when cofactors stabilise the unstable structure of anthocyanins. The cofactors nature is wide; compounds such as flavonoids and non-flavanoids polyphenols, amino acids and organic acids may act as cofactors (Darias-Martin et al. 2001). At the same time, acetaldehyde and pyruvate released out of the yeast cytoplasm may produce vitisins from the condensation with anthocyanins; this process varies as a function of the yeast strains used (Suárez-Lepe and Morata 2012). Yeasts like Saccharomyces cerevisiae and some nonSaccharomyces yeasts like Pichia guillermondii have hydroxycinnamate decarboxylase activity; this enzymatic activity maximises the production of vinyl phenolic pyranoanthocyanins from the chemical interaction between hydroxycinnamic acids and anthocyanins (Morata et al. 2012). Flavanols are flavonoids naturally found in grape skins; these polyphenols, once extracted into the must, may interact with grape anthocyanins through direct bonding or through ethyl bridges (Mateus et al. 2016) during alcoholic fermentation and wine aging stimulating the production of more stable pigments as a result of those chemical changes (Zeng et al. 2015). Model solutions have been used to describe the mechanisms in which flavanol oligomers are condensed with anthocyanin moieties (Salas et al. 2004) while other studies show the influence that the yeasts have in the formation of stable pigments such as vitisins, vinylphenolic pyranoanthocyanins and flavanol-anthocyanins adducts (Morata et al. 2016). In this last matter, the selection of yeasts, seeking for the formation of stable pigments, will probably also affect the aromatic profile of wines through the formation of

3

higher alcohols and esters during fermentation; this is the case for the yeast species Torulaspora delbrueckii that may enrich the aroma of wines or the yeasts species Kloeckera, Hanseniospora, Candida, Pichia and Metschnikowia that increase the amount of metabolic esters (Suárez-Lepe and Morata 2012). The possibility of having more pigment precursors may increase the formation of polymeric moieties by carrying out mixed or sequential fermentations. A sequential fermentation will countervail the relatively low fermentative power of most non-Saccharomyces yeasts to reach a higher ethanol content and to boost both the formation of stable pigments and the aromatic profile by using yeasts Saccharomyces cerevisiae (Benito et al. 2015, Loira et al. 2014) and Schizosaccharomyces pombe (Benito et al. 2012, Suárez-Lepe et al. 2012). The sequential fermentation using Saccharomyces and Schizosaccharomyces yeasts species would allow us to evaluate the influence of both yeasts in the colour evolution through the characterization of pigments. Therefore the aim of this work was to compare nonSaccharomyces yeasts as enhancers of the formation of polymeric pigments; nonSaccharomyces yeasts acting as promoters of the production of precursor compounds from metabolism origin. In the mean time, the evolution of yeast metabolites that will influence the aroma profile of these wines was also followed.

2. Materials and Methods 2.1 Yeast strains and growing media

4

Three non-Saccharomyces yeasts (Lachancea thermotolerans (Lt) strain CBS 2803, Metschnikowia pulcherrima (Mp) strain LAMAP-USACH L1781 and Torulaspora delbrueckii (Td) strain LYCC 7013) were used in sequential fermentation with Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp) strain 938 (IFI 938). The yeasts Lt, Mp and Td were grown in stripe and liquid YPD media during 72 h at 25 ºC to reach a population in the pre-inoculum of 9.05 X 108, 1.04 X 109 and 8.40 X 108 CFU/mL by count plates respectively. The growth of the species Sc and Sp was carried out in same liquid YPD media at 25 ºC during 48 h to reach a population of 2.70 X 108 and 1.85 X 108 CFU/mL in the respective pre-inoculum. The liquid YPD media was prepared with 1 % yeast extract (Laboratorios Conda; Madrid, Spain), 2 % bacteriological peptone (Laboratorios Conda; Madrid, Spain) and 2 % D(+)glucose anhydrous (Panreac Química; Barcelona, Spain); liquid YPD media was sterilized in autoclave at 120 º C during 15 min.

2.2 Red must fermentation Red grapes must, Vitis vinifera L. cv. Tempranillo from the vintage 2012 was used. The must had density of 1101 g/dm3 and pH of 3.5 after adding 2.4 g of L(+)-tartaric acid (Panreac Química; Barcelona, Spain). The alcohol potential for this must was 14.1 %v/v. The must was sterilized in autoclave at 100 ºC during 1 min in 1 L containers. Microbiological control analysis showed no presence of yeast colonies (100 in 100 µl) and 7 FCU/mL of mesophilic bacteria in the must prior the inoculation of non-Saccharomyces yeasts.

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The sterile must was divided into four different treatments in conical flasks with 900 mL (3 flasks) and one with 150 mL. The treatments were named Control, (+)-Catechin, ProcyanidinB2 and ProcyanidinC1 after the flavanol used during the fermentation. The flavanols (+)-catechin (CAS: 154-23-4 and purity of 99 %), procyanidinB2 (CAS: 2910649-8 and purity of 90 %) and procyanidinC1 (CAS: 37064-30-5 and purity of >95 %) (Cymit Química S.L., Barcelona, Spain) were used at a constant concentration of 40 mg/L. The musts were divided into 60 mL and 10 mL microfermenters for fermentation volumes of 50 and 10 mL and then added with 750 µL and 120 µL of inoculum of each nonSaccharomyces medium fermentative yeast strain respectively and by triplicate. Microfermenters were sealed with Müller valves and placed at constant 25 ºC after being weight. After the 5th day a second inoculum with yeasts Sc or Sp was added to all trials to finish fermenting residual sugars and to achieve higher alcohol volume. The fermentation kinetics was followed until steady weight was reached. Fermentations spanned up to 21 days. The experiment comprised the following trials: each of the three non-Saccharomyces yeasts did sequential fermentations with both Sc and Sp. This resulted in 6 different fermentation trials (Lt-Sc, Mp-Sc, Td-Sc, Lt-Sp, Mp-Sp and Td-Sp) by triplicate for each of the four different treatments (Control, (+)-Catechin, ProcyanidinB2 and ProcyainidinC1).

2.3 Volatile compounds analysis Volatile compounds were characterized by gas chromatography with flame ionization detector (GC-FID). The equipment used was an Agilent Technologies™ 6850 (Palo Alto,

6

CA, USA) with a column DB-624 (60 m x 250 µm x 1.4 µm). The injector’s temperature was 250 ºC and the detector’s temperature was set to 300 ºC. The temperature went from a steady 40 ºC for 5 min to 250 ºC with a gradient of 10 ºC/min. This temperature was then maintained for 5 min. Hydrogen was used as carrying gas at a 2.2 L/min flow with a split ratio of 1:10. Identification and quantification of volatile compounds were performed with 100 µL of 4-methyl-2-pentanol (500 mg/L) used as an internal standard (Abalos et al. 2011).

2.4 Infrared spectroscopy The equipment OenoFoss™ (FOSS Iberia, Barcelona, Spain) using Fourier transform infrared spectroscopy (FTIR) was used to identify and quantify major compounds in wine such as ethanol and other common compounds like malic and lactic acid, volatile acidity, glucose and fructose. This technique also determines pH values.

2.5 Pigments characterization HPLC with diode array detector and electro spray ionization coupled to mass spectroscopy (DAD-ESI/MS)

has

been

used

to

identify

and

characterize

anthocyanins,

pyranoanthocyanins and polymeric pigments; an Agilent Technologies™ 1100 (Palo Alto, CA, USA) chromatograph with a column RP Kinetex C18 (100 x 4.6 mm; 2.6 µm) (Phenomenex, Torrance, CA, USA) was used for this purpose. Two solvents were used: solvent A (water/formic acid 95:5 v/v) and solvent B (methanol/formic acid 95:5 v/v) with

7

the following gradient of solvent B (0.8 ml/min): from 20 % to 50 % from time 0 to 27 min; 50 % from time 27 to 28 min and finally from 50 % to 20 % from time 28 to 29 min until reaching a steady state. According to Loira et al. (2014) the malvidin-3-O-glucoside has been used as an external standard at a wavelength of 525 nm for the quantification of all pigments while the identification was carried out with mass spectrometry positive scanning from 100 to 1500 m/z from time 0 to 23 min. Detection limit was set to 0.1mg/L (Morata et al. 2016).

2.6 Colour determination The colour of red wine has been determined by the use of a UV-visible (UV-Vis) spectrophotometer 8453 from Agilent Technologies™ (Palo Alto, CA, USA) with a photodiode array detector and the use of a 1mm path length cuvette. The absorption at three different wavelengths (420, 520 and 620 nm) was used to compare colour intensity and hue in all wines after fermentation was complete. The results are also expressed as the percentage of yellow, red and blue fractions present in each finished wine, obtained from the different absorption signals (Kulkarni et al. 2015).

2.7 Sensory evaluation The sensory analysis of the wines produced (except for the treatment ProcyanidinC1 due to short volume produced) was assessed with a panel of 10 experts in wine tasting, both genders and ages from 28 to 54 and without the use of a standard kit. The panel was

8

constituted by members of the Chemistry and Food Technology department of the School of Agricultural, Food and Biosystems Engineering at Technical University of Madrid (Spain) with over 5 years experience in wine tasting. 14 attributes, basic wine descriptors previously agreed by consensus, were rated with a five-point scale from low perception (1) to high perception (5) except for the hue that was rated from red – young wines (1) to orange – aged wines (5). The attributes evaluated were: colour intensity, hue, aroma intensity, aroma quality, herbs, flowers, fruitiness, reduction, oxidation, body, astringency, bitterness, general acidity and overall impression.

2.8 Statistical analysis Means and standard deviations were calculated and differences examined using ANOVA and the least significant difference (LSD) test. All calculations were made using PC Statgraphics v.XI software (Graphics Software Systems, Rockville, MD, USA). Significance was set at P<0.05.

3. Results and discussion 3.1 Wine composition There is a slight, yet not significant, lower ethanol content for those fermentations done with Mp and Td in sequential fermentation with Sp (12.1-12.3 % v/v vs. >12.5 % v/v). The pair Mp-Sp had the lowest malic acid content (0.3-0.4 g/L) and therefore the highest pH value (3.9) in all treatments and the lowest total acidity value in each treatment (2.3-2.6

9

g/L).

Suárez-Lepe

et

al.

(2012)

have

documented

the

demalication

of

Schizosaccharomyces spp. through which malic acid is transformed into ethanol and CO2. The lactic acid is present in small amounts in wines fermented with Mp-Sp (0.1 g/L) with significant difference in the Control and the (+)-Catechin treatment, while the other treatments with Mp had smaller amounts of lactic acid with no significant difference with the rest of the trials. In these trials, despite the results reported by Ciani et al. (2010), the fixed acidity (mainly L-lactic acid) was not high for fermentations where Lt was used. The amount of residual sugars is significantly higher for the sequential fermentation done with Td-Sp for the treatments ProcyanidinB2 and ProcyanidinC1 (5 g/L and 7 g/L respectively).

3.2 Fermentative volatile compounds Regarding the formation of ethyl acetate, a compound responsible of solvent or glue aroma in wines when in high concentration (Peinado et al. 2004), it can be seen that all pairs of fermentative yeasts having Mp had concentrations larger than 125 mg/L; the pair Mp-Sp had the largest maximum values with significant difference in all treatments with values as high as 280 mg/L for the treatment ProcyanidinC1 (Table1). These values are considerably high compared to a single culture fermentation involving Sc producing approximately 4.7 mg/L or a sequential fermentation involving different strains of Td and Sc with values between 6 mg/L and 30 mg/L according to Loira et al. (2014). Compounds such as acetaldehyde, acetoin and diacetyl are also shown in Table1. Regarding the amount of acetaldehyde it can be seen that the pair Mp-Sp had significant maximum values (above 30 mg/L) for the control and the treatments (+)-Catechin and

10

ProcyanidinB2; the pair Td-Sc had the largest value for the treatment ProcyanidinC1 (>70 mg/L). The values for acetaldehyde concentration are in general larger for all the trials in the treatment ProcyanidinC1 than the other treatments. In terms of acetoin content, the pair Mp-Sp also had significant maximum values for the control and the treatments (+)-Catechin and ProcyanidinB2; the values reached are close to the threshold (10mg/L) (Benito et al. 2011) and are similar to the results observed for the pair Lt-Sp in treatment ProcyanidinC1. Finally, the diacetyl was found to have significant maximum values only for the treatment ProcyanidinC1 for both trials having Mp while there is a significant minimum value for the trial Td-Sp for treatment ProcyanidinB2. The threshold for the diacetyl in wines is 2 mg/L according to Benito, S. et al. (2011) and several trials are on the upper threshold level or even above it. The concentration of total esters had the same tendency as the ethyl acetate concentration. The trials where the Mp was used have significant maximum values. The pair Mp-Sp produced the largest amount of esters (299 ± 8 mg/L) in treatment ProcyanidinC1. Other esters like ethyl lactate and 2-phenyl ethyl acetate were found to be statistically more abundant in trials with the yeasts Lt-Sp. The control and the treatment ProcyanidinB2 had larger concentrations of ethyl lactate (between 7.5 and 8.8 mg/L) while the treatment ProcyanidinC1 had larger amount of 2-phenyl ethyl acetate (23.3 ± 0.7 mg/L). Even though some esters could induce odd aromas when overpassing the threshold levels, ethyl lactate produces coffee or strawberry and raspberry aromas (Vilanova et al. 2010) and 2phenylethyl acetate smells like roses, honey or apples (Duarte et al. 2013). The overall all esters may contribute to fruity and flower aromas (Boss et al. 2015) and therefore any of the potential aroma defects were detected in these wines during the sensory evaluation.

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Regarding higher alcohols, among the statistical maximum values for each treatment, the amount obtained in treatment ProcyanidinC1 for trial Mp-Sp reached 491 ± 7 mg/L, which also correlates to the large value obtained for 2-phenyl ethyl alcohol for this trial (85 ± 2.4 mg/L). The concentration of higher alcohols is close to the threshold 350 mg/L and it could be aromatic defect in wines (Rapp and Mandery 1986, Rapp and Viersini 1995) in some cases. At last, the amount of 2,3-butanediol had statistical maximum values for trials having Mp as fermentative yeast while trials where Lt was used the concentration of this alcohol had minimum values in all four treatments. 2,3-butanediol may contribute to enhance organoleptic properties in trials with Mp since this compound has neutral aroma but sweetens and softens the wine (Jackson 2008, Romano et al. 1998). The concentration of total volatiles follows the trend observed for ethyl acetate, 2,3butanediol and 2-phenyl ethyl alcohol where Mp was used as fermentative yeast; larger values were observed for this species with disregard of the yeast used for the sequential fermentation or the treatment. On the other hand, the use of Td in the control treatment lead to lower volatiles concentration values while, Lt produced less total volatiles in all three treatments where the flavan-3-oles where added. A PCA was done for the eleven volatile compounds plot in Figure1. The distribution is explained by the first two components. PC1 is positively contributed by total volatiles, esters and ethyl acetate and, negatively contributed by ethyl lactate and 2-phenyl ethyl acetate. PC2 is positively contributed by acetaldehyde and higher alcohols and, negatively contributed by 2,3-butanediol. Four different clusters can be identified after PCA. Regarding PCA1 the fermentative non-Saccharomyces yeasts are highlighted and clusters are split into trials fermented with Mp with higher concentration of ethyl acetate and esters

12

while trials fermented with Td and Lt are explained by the variables ethyl lactate and 2phenyl ethyl acetate. Regarding PC2 the distribution highlights the treatment being the Control and ProcyanidinC1 –where polymeric pigments are scarce (ProcyanidinC1) or null (Control)– influenced by the acetaldehyde and higher alcohols concentration while treatments (+)-Catechin and ProcyanidinB2 are influenced by 2,3-butanediol.

3.3 Anthocyanins characterization The amount of monomeric anthocyanins including the non-acylated, and the acetyl-, pcoumaroyl- and caffeoyl derivatives, vary in function of the treatment and the yeasts used in the sequential fermentations. It can be seen that the treatment ProcyanidinC1 kept more non-acylated pigments over the fermentation comparing same yeast pairs between treatments. If we consider the pair Lt-Sp, this trial had the highest concentration of nonacylated anthocyanins at the end of the fermentation with 74.1±0.7 mg/L with significant difference to all other results but considerably low compared to the initial 172.2±1.3 mg/L found in the must. Regarding the rest of monomeric derivatives it can be seen that the trials with highest amount of pigments are the pair Td-Sc for the acetyl derivatives in the treatment ProcyanidinC1 and the same pair for the p-coumaroyl and caffeoyl derivatives in the treatment ProcyanidinB2. In terms of total anthocyanins, ProcyanidinC1 is consistently having the highest concentration among all treatments for each specific pair of yeasts. The single trials having the highest amounts of total anthocyanins with no significant difference were fermented with the yeasts Lt-Sc, and Lt-Sp (Figure2).

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3.4 Vitisins and vinyl phenolics In terms of vitisins formation, the trials having higher concentration of these pyranoanthocyanins had fermented with Lt-Sc in treatment ProcyanidinC1 and with Td-Sp in treatment ProcyanidinB2 (both 1.3±0.0 mg/L) followed by the same yeast pairs but in the treatments (+)-Catechin and Control respectively (both 1.1±0.0 mg/L) (Table2). The vitisins identified with the mass spectroscopy are malvidin-3-glucoside pyruvate (Vitisin A) and malvidin-3-glucoside acetaldehyde (Vitisin B) with molecular ion [M]+ (m/z) 561 and fragment ion with (m/z) 399 and [M]+ (m/z) 517 and fragment ion with (m/z) 355 (Vergara et al. 2010) respectively; malvidin-3-(6''-acetylglucoside) pyruvate and malvidin3-(6''-acetylglucoside) acetaldehyde were identified with [M]+ (m/z) 603 and fragment ion (m/z) 399 and [M]+ (m/z) 559 and fragment ion (m/z) 355 (He et al. 2012) respectively. The Control as well as the treatment (+)-Catechin reported the presence of vitisin A and vitisin B; the treatment ProcyanidinB2 had vitisin A, vitisin B and malvidin-3-(6”acetylglucoside) pyruvate while the treatment ProcyanidinC1 had, besides vitisin A and vitisin B, malvidin-3-(6”-acetylglucoside) pyruvate in the trials fermented with Lt-Sc and malvidin-3-(6”-acetylglucoside) acetaldehyde in the trial fermented with Td-Sc. The vinyl phenolic compounds identified were the malvidin-3-glucoside-4-vinylphenol and malvidin-3-glucoside-4-vinylguaiacol identified with the molecular ions [M]+ (m/z) 609 and 639 respectively (He et al. 2012). The pair Mp-Sc had the highest concentration of vinyl phenolics in all treatments and out of these ones the trial from the treatment ProcyanidinB2 is the one with the highest value (0.15±0.0 mg/L). According to Suárez-

14

Lepe

and

Morata

(2015),

non-Saccharomyces

yeasts

with

hydroxycinnamate

decarboxylase activity (HCDC) such as Sp and Torulaspora spp. among other species produced the decarboxylation of hydroxycinnamic acids into vinyl phenols. Despite these findings, there was no trace of vinyl phenolic compounds in the trials fermented with Td-Sp in any of the treatments and in the specific trials Lt-Sc in the control and Lt-Sp in the treatment ProcyanidinC1.

3.5 Polymeric pigments The intervention of acetaldehyde in the condensation mechanism for the formation of polymeric pigments with a pronounced effect in the presence of flavanols (Dallas et al. 1996) is confirmed through the analysis of stable pigments especially in treatments (+)Catechin and ProcyanidinB2. The dimers formed with added (+)-Catechin were identified as malvidin-3-glucoside-ethyl-catechin enantiomers with molecular ion [M]+ (m/z) 809 and fragment ions (m/z) 657, 331 (He et al. 2012, Monagas et al. 2003) and retention times of 10.5 min and 11.2 min. Both UV-Vis signals had a shoulder between wavelengths 450 nm and 460 nm that correlates with Morata et al. (2016) observations for ethyl linkages for indirect condensation of flavanols with anthocyanins. The yeasts Lt-Sp yielded the largest amount of dimeric polymers. In general for treatment (+)-catechin, the trials were the yeast Sp was used for sequential fermentation had produced more polymeric pigments than its counterpart fermented with Sc. Regarding the formation of polymers with procyanidin B2, the trials fermented with Sp as sequential fermentative yeast had larger concentration values of polymers. The compound

15

identified in all these trials is malvidin-3-O-glucoside-ethyl-procyanidinB2 with molecular ion [M]+ (m/z) 1097 and fragment ion (m/z) 519 (He et al. 2012, Morata et al. 2016). The shoulder observed in the UV-Vis signal for anthocyanin-flavanol ethyl linkage at 460 nm was not sharp for these polymeric compounds as it was for the (+)-catechin enantiomers. The trial Td-Sp registered the highest value of this polymeric pigment with 0.33±0.0 mg/L. The evidence of the formation of polymers by condensation of procyanidin C1 with anthocyanins was not possible to assess since the concentration may be scarce to distinguish single peaks in the chromatograms and the signal from the UV-Vis and the mass spectrometer had relatively high noise signal for such concentration level. Few peaks were indicated at retention time 10.6 min with (m/z) 1187 as potential non-identified oligomer condensate products. These products had a shoulder signal at wavelength 460 nm similar to that produced by an ethyl linkage previously observed in other anthocyaninflavanol condensates. Nonetheless, statistically speaking, there is no significant difference in treatment ProcyanidinC1 with respect to the control with any polymeric pigments at all.

3.6 Colour assessment The colour intensity as well as the hue were determined for all the sequential fermentations in all treatments and, as it can be seen in Table3A, the fermentations done with added procyanidin B2 had higher colour intensity values with no significant difference among the trials within the same treatment; the trials in the control set had significant difference between the sequential fermentations done with Sc and those done with Sp, the trials fermented with Sc had lower colour intensity values. The values of the total polyphenol

16

index (TPI) had the same profile than the colour intensity with the highest values obtained with the addition of procyanidin B2 (Table3C). On the other hand, there are no significant difference in hue values in the trials of all treatments except for the fermentations done with Mp-Sp where, in all treatments, had the highest values with significant differences in each treatment (Table3B).

3.7 Wine sensory evaluation Most of the vinification trials did not have statistical differences in the descriptive analysis except for those attributes highlighted with letters “a” and “b” on the Figure3. The spider net charts show the attributes with significant difference where the panel evaluated wines differently. These attributes correspond to the aroma quality, the fruitiness and the overall impression. The trial Td-Sc in the control was rated with the highest score for the attributes aroma quality and fruitiness with statistical maximum values; the trial Mp-Sc for treatment ProcyanidinB2 was attributed the lowest score for aroma quality which, regarding volatile compounds analysis, it may correspond to the rather large amount of diacetyl, ethyl acetate and 2-phenyl ethyl alcohol (Table1). The overall impression of the trial Td-Sp obtained the highest score for the treatment ProcyanidinB2; this trial had some of the lowest concentrations of total esters measured including the low amount volatiles responsible of wine faults. Out of the results obtained, especially the impressions obtained for the trial Mp-Sc differ from the experience obtained by Sadineni et al. (2012) where both nonSaccharomyces yeasts were compared after fermenting mango wine and both obtained better scores whilst the wine fermented with pure Sc scored worse; in terms of preference

17

González-Royo et al. (2014) showed that 6 out of 9 tasters identified single culture Sc fermentation from sequential fermentation with Td and 5 of these 6 tasters that noted the difference preferred the sequential fermentation wine. Sun et al. (2014) found that nonSaccharomyces yeast reinforced sweet, green and fatty notes and improved fruity odour. The results shown in this work compared the effect of these three non-Saccharomyces yeasts in sequential fermentations establishing Td as the one improving the attributes of red wines in general. Any negative effect attributed to procyanidins addition was detected in the sensory evaluation.

4. Conclusions The formation of polymeric pigments may be influenced not only by the availability and nature of the flavanol itself, where (+)-catechin seemed to have better affinity to condensing with anthocyanins, but also by the synergistic effect with the yeasts during the sequential fermentation. It is, through the sequential fermentation that the production of precursors for the formation of pyranoanthocyanins such as vitisins and vinyl phenolic compounds may differ from single culture fermentations. In this matter, the use of yeasts capable of enhancing the production of acetaldehyde may play an important role on the formation of such stable pigments; these key factors are currently under study in model solutions where non-Saccharomyces yeasts are evaluated. Finally, it can be said that the results observed in this study strengthen the fact that non-Saccharomyces yeasts are prone to modifying aromatic profile in red wines as well as stabilising pigments through the formation of metabolic precursor compounds.

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Conflict of Interest All authors declare not to have conflict of interest.

Acknowledgements Special thanks to Consejo Nacional de Ciencia y Tecnología (CONACYT – México) for the pre-doctoral scholarship granted and to all personnel from Department of Chemistry and Food Technology in the ETSIAAB at Technical University of Madrid for the support received.

Funding: This work was supported by Ministerio de Economía, Industria y Competitividad (MINECO – Spain) [grant number AGL2013-40503-R].

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

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23

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24

time‐of‐flight

mass

spectrometry.

Rapid

Communications

in

Mass

Spectrometry, 30(1), 81-88.

Figure captions. Figure1. Projection of the 72 wine trials on the first two components of the PCA. Identification of clusters formed: 1) Control and ProcyanidinC1 (Lt and Td); 2) Control and ProcyanidinC1 (Mp); 3) (+)-Catechin and ProcyanidinB2 (Lt and Td); 4) (+)-Catechin and ProcyanidinB2 (Mp). Figure2. Plot to compare polymeric pigments and total pigments after fermentation. Trials are grouped by treatment; diamonds for control, squares for (+)-catechin, triangles for procyanidin B2 and circles for procyanidin C1. Yeasts producing larger levels of total pigments are shown in italic black characters; yeasts forming the larger amounts of polymeric pigments are shown in underlined italic grey characters. Figure3. Spider net chart for taste panel results for wine sensory evaluation: 1) Control, 2) (+)-Catechin and 3) ProcyanidinB2. Series with letters indicate significant difference between means (p < 0.05).

25

PC2 (17.88%)

3.7

1

Acetaldehyde

Lt-Sp-PC1 Lt-Sp-C

2.7

Higher alcohols Lt-Sp-PC1 Lt-Sp-C

Lt-Sc-PC1 Lt-Sc-C Lt-Sp-PC1 Lt-Sp-C Td-Sc-PC1 Td-Sc-C Td-Sc-PC1 Td-Sc-C Lt-Sc-PC1 Lt-Sc-C Lt-Sc-PC1 Lt-Sc-C Td-Sc-PC1 Td-Sc-C

2-phenylethyl acetate

1.7

2-phenylethyl alcohol

2

Ethyl lactate

0.7

Mp-Sc-PC1

Td-Sp-C Lt-Sc-CA Lt-Sc-CA

-0.3

Td-Sp-PC1 Td-Sp-C Td-Sp-C Td-Sp-PC1

Lt-Sp-CA Lt-Sc-PB2 Td-Sp-CA Lt-Sp-CA Lt-Sp-PB2 Lt-Sc-CA Lt-Sp-CA Td-Sp-PB2 Td-Sc-PB2 Lt-Sc-PB2 Lt-Sp-PB2 Lt-Sc-PB2

-1.3

Td-Sc-CA

Mp-Sp-PB2

Mp-Sp-PB2

Mp-Sc-CA Mp-Sc-PB2

4

Mp-Sc-PB2 Mp-Sp-CA

Mp-Sp-CA

-1

Total Esters Ethyl acetate

Mp-Sc-CA Mp-Sc-PB2

Td-Sc-PB2

3 -3

Mp-Sc-PC1 Mp-Sc-C

Mp-Sc-CA

Mp-Sp-CA

-2.3

Mp-Sp-C

Mp-Sc-C

Acetoin

Td-Sp-PB2 Td-Sc-CA Td-Sp-CA Td-Sc-PB2 Td-Sp-CA Td-Sc-CA

Mp-Sp-PC1 Mp-Sp-C Mp-Sp-PC1

Mp-Sc-PC1

Lt-Sp-PB2

Mp-Sp-C Mp-Sp-PC1

Mp-Sc-C

Diacetyl

Td-Sp-PC1

1

Mp-Sp-PB2

Butanediol

3

5

7

PC1 (44.75%)

Total pigments (mg/L)

90

Procyanidin C1 Lt-Sp

80 Lt-Sc

Lt-Sc Lt-Sc

70

Lt-Sp

Lt-Sp Mp-Sp

Procyanidin B2

(+)-Catechin

60

Control 50 0

0,2

0,4

0,6

0,8

1

Polymeric pigments (mg/L)

Overall impression

Color Intensity 4

Color Intensity 4

Overall impression

Hue

3

3

Aroma Intensity

Acidity

2

1

1

Bitterness

Aroma Quality

Astringency

Astringency

Herbs

Body

Oxidation Reduction

MpOverall - Sp

Lt - Sp

Flowers

Oxidation

Fruitiness Td - Sc

Herbs

Body

Flowers

Mp - Sc

Aroma Quality

0

0

Lt - Sc

Aroma Intensity

Acidity

2 Bitterness

Hue

impression

Color Intensity Lt - Sp4

Fruitiness Reduction

Lt - Sc

Hue

Mp - Sc

3

Td - Sc

Aroma Intensity

Acidity 2

Aroma Quality

1

Bitterness

0 Astringency

Herbs

Body

Flowers

Oxidation Lt - Sc

Mp - Sc

Fruitiness Reduction Td - Sc Lt - Sp

Mp - Sp

Td - Sp

Lt - Sp

Mp - Sp

Lt - Sp

Table1. Concentration of volatiles from fermentative origin in finished wines [mg/L]. Mean ± STD (n=3). Yea sts

Acetalde Diacet hyde yl

Ethyl acetate

Acetoi n

Ethyl lactate

45 ± 8c

6.9±0. 2b 7.3±0. 1b 6.7±0. 1b 6.9±0. 3b 10.5± 2.7a 7.8±0. 2b

6.9±0.4a

2,3butanedi ol

22phenyle phenyl thyl ethyl alcohol acetate

Esters*

Higher alcohols**

60 ± 9c

401 ± 31a

157 ± 20b

362 ± 4ab

103 ± 5c

314 ± 5b

Total

Control LtSc MpSc TdSc LtSp MpSp TdSp

14 ± 2c 21 ± 1b 14 ± 1c 12 ± 1c 31 ± 2a 21 ± 3b

2.9 ± 0.8a 2.9 ± 0.6a 3.3 ± 0.2a 2.2 ± 0.2a 3.1 ± 0.1a 2.7 ± 3.1a

134 ± 18b 84 ± 5bc 54 ± 4c 204 ± 65a 47 ± 9c

b

6.0±0.0a b

6.0±0.1a b

502 ± 43c 58 ± 5a 788 ± 34a 61 ± 1a 557 ± 16b 31 ± 2b

8.2±0.5a 510 ± 1bc 6.3±0.1a b

838 ± 21a

4.5±3.9b

553 ± 37bc

45 ± 10ab 59 ± 21a 35 ± 2b

5.4 ± 0.1a 6.4 ± 0.2a 5.3 ± 0.0a 5.2 ± 0.1a 5.7 ± 0.3a 7.6 ± 3.9a

1,002 ± 72b 1,354 ± 55a 1,013 ± 15b

67 ± 5c

359 ± 34ab 972 ± 32b

223 ± 62a

363 ± 90ab

1,484 ± 171a

59 ± 9c

304 ± 13b

962 ± 57b

64 ± 9b

369 ± 38a 986 ± 41bc

160 ± 21a

360 ± 9a

(+)-Catechin LtSc MpSc TdSc LtSp MpSp TdSp

15 ± 1bc 19 ± 1b 13 ± 1c 13 ± 1c 31 ± 5a 18 ± 6bc

2.2 ± 0.8ab 2.0 ± 0.1ab 2.2 ± 0.1ab 1.5 ± 1.3ab 2.5 ± 0.3a 0.8 ± 1.4b

50 ± 8b 138 ± 20a 70 ± 14b 44 ± 1b 136 ± 39a 47 ± 11b

6.7±0. 0b 7.0±0. 4b 6.8±0. 2b 6.7±0. 1b 9.7±2. 7a 7.3±0. 4b

6.6±0.3a 515 ± 16d

51 ± 10b

6.2±0.2a 784 ± 44b 62 ± 1a 2.0±3.5b 565 ± 40c 35 ± 5d 7.8±0.2a

512 ± 8d

41 ± 2cd

5.9±0.1a 863 ± 18a 47 ± 1bc 6.4±0.1a

606 ± 8c

36 ± 3d

5.2 ± 0.1ab 6.5 ± 0.1ab 3.5 ± 3.0b 5.2 ± 0.2ab 5.7 ± 0.4ab 8.6 ± 5.8a

80 ± 15b 333 ± 26ab

1,347 ± 33a 1,015 ± 66b

58 ± 1b

338 ± 9ab

943 ± 10c

153 ± 42a

302 ± 13b

1,376 ± 46a

63 ± 7b

313 ± 5b

1,024 ± 5b

Procyanidin B2 LtSc MpSc TdSc LtSp MpSp TdSp

15 ± 1c 21 ± 2b 15 ± 1c 15 ± 2c 34 ± 3a 26 ± 6b

2.2 ± 0.2abc 2.4 ± 0.2ab 2.5 ± 0.2ab 1.9 ± 0.2bc 3.0 ± 0.2a 1.3 ± 1.2c

48 ± 2d 128 ± 9b 63 ± 5c 44 ± 2d 166 ± 12a 43 ± 2d

6.7±0. 1b 7.1±0. 3b 7.0±0. 0b 6.8±0. 1b 13.9± 6.8a 8.3±0. 6b

7.5±0.5a b

6.2±0.1a b

512 ± 23c 39 ± 5b 846 ± 34a 57 ± 4a

2.0±3.5c 571 ± 20b 33 ± 1b 8.8±1.4a 560 ± 8bc 6.1±0.1a b

4.4±3.8b c

879 ± 42a

36 ± 10b 57 ± 12a

563 ± 29b 31 ± 3b

5.2 ± 0.1c 6.4 ± 0.1a 5.2 ± 0.0c 5.2 ± 0.1c 5.7 ± 0.2b 5.6 ± 0.3b

63 ± 3cd 327 ± 32ab

941 ± 7d

148 ± 8b

340 ± 4ab

74 ± 3c

337 ± 10ab

1,380 ± 40b 1,023 ± 14c

58 ± 4d

300 ± 50b

959 ± 38d

185 ± 12a

367 ± 47a

1,497 ± 38a

56 ± 4d

302 ± 5b

973 ± 32cd

Procyanidin C1 LtSc MpSc TdSc

52 ± 10b 28 ± 5c 74 ± 12a

2.3 ± 0.2b 3.6 ± 0.7a 2.3 ± 0.1b

47 ± 4cd 196 ± 15b 56 ± 2c

7.0±0. 6.8±0.2a 486 ± 30d 62 ± 5c 1b 6.8±0. 4.3±3.7a 619 ± 11b 76 ± 8b 2b 7.4±0. 6.2±0.2a 535 ± 3c 45 ± 2d 1b

5.3 ± 0.1c 5.9 ± 0.2b 5.2 ± 0.0c

62 ± 4cd 476 ± 13ab 214 ± 13b

445 ± 48bc

70 ± 2c

425 ± 2cd

1,100 ± 53cd 1,332 ± 58b 1,130 ± 13c

26

LtSp MpSp TdSp

51 ± 12b 45 ± 3b 37 ± 8bc

2.5 ± 0.4b 3.3 ± 0.3a 2.1 ± 0.2b

43 ± 2d 285 ± 8a 46 ± 4cd

9.4±1. 6.6±0.4a 488 ± 30d 58 ± 5c 3a 7.4±0. 6.2±0.1a 734 ± 41a 89 ± 2a 1b 7.3±0. 6.3±0.2a 603 ± 11b 43 ± 1d 7b

23.3 ± 0.7a 5.3 ± 0.1c 5.3 ± 0.1c

73 ± 2c

403 ± 15d

299 ± 8a

491 ± 7a

57 ± 4d

394 ±10d

1,042 ± 54d 1,595 ± 32a 1,117 ± 23cd

Different letters in the same column indicate significant differences between means (p<0.05). Treatments are analysed separately. *

Esters account for the sum of: ethyl acetate, ethyl lactate, 2-phenylethyl acetate, isoamyl acetate and ethyl butyrate

**

Higher alcohols account for the sum of: 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isobutanol, propanol, butanol, hexanol and 2-butanol

27

Table2. Concentration of pigments in wines and must expressed as [mg/L]. Polymeric pigments include adducts formed with flavanols. Mean ± STD (n=3).

Yeast s

Non Acylate d1

Must

172.2± 1.3a

Anthocyanins pAcetylgluco Caffeoylgluco Coumaroylgluc 2 sides sides4 3 osides

9.4±0.1a

Pyranoantocyanins 5

Vitisins

Vinylphen olics6

Polyme ric pigmen ts7

Total pigment s

12.6±0.1a

0.9±0.0a

0.00±0. 00k

0.00±0.00

0.00±0. 00k

195.1± 1.4a

5.1±0.0fgh

0.7±0.0bcd

0.00±0.00

5.1±0.0fgh

0.7±0.0bcd

5.1±0.0fgh

0.6±0.0cdef

4.7±0.0jkl

0.6±0.0hijk

5.1±0.0fgh

0.6±0.0defgh

4.3±0.1m

0.5±0.0klm

0.89±0. 04efg 0.97±0. 01d 0.89±0. 01efg 1.09±0. 03bc 0.83±0. 00ghi 1.13±0. 01b

0.00±0. 00k 0.00±0. 00k 0.00±0. 00k 0.00±0. 00k 0.00±0. 00k 0.00±0. 00k

72.9±0. 4def 66.3±0. 6jkl 61.2±0. 1no 70.5±0. 6fgh 55.6±0. 6pq 63.8±0. 4lmn

5.0±0.2hi

0.6±0.0bcde

5.2±0.1efg

0.6±0.0defg

5.3±0.2def

0.6±0.0defg

4.6±0.1l

0.5±0.0jklm

5.0±0.0gh

0.6±0.0ghij

4.7±0.2ijkl

0.5±0.0lm

1.13±0. 0.05±0.00 hi 03b 0.79±0. 0.12±0.00 b 02hi 0.84±0. 0.08±0.00 e 02fgh 1.05±0. 0.07±0.00f g 03c 0.80±0. 0.10±0.01 d 03hi 1.10±0. 0.01±0.00 k 02bc

0.64±0. 01c 0.46±0. 01e 0.44±0. 02e 0.81±0. 01a 0.52±0. 02d 0.74±0. 00b

75.6±2. 3cd 65.0±0. 6klm 62.5±1. 2mn 68.7±0. 6hij 54.6±0. 3r 66.0±1. 2jkl

5.5 ± 0.0bcd

0.7±0.0bc

5.7 ± 0.0bc

0.7±0.0b

5.7 ± 0.0b

0.7±0.0b

4.9 ± 0.0hij

0.6±0.0fghij

5.2 ± 0.1fgh

0.6±0.0efghi

4.2 ± 0.0m

0.5±0.0m

0.90±0. 0.05±0.00 hi 02ef 0.93±0. 0.15±0.00 a 03de 0.98±0. 0.06±0.00 gh 02d 1.06±0. 0.03±0.00j 02c 0.83±0. 0.08±0.00 e 02ghi 1.28±0. 0.00±0.00 k 02a

0.09±0. 00j 0.25±0. 00h 0.20±0. 00i 0.28±0. 00g 0.22±0. 01i 0.33±0. 00f

74.2±0. 5cde 69.3±1. 2ghi 68.8±1. 3hij 72.7±1. 4def 58.2±1. 2op 67.5±0. 4ijk

4.7±0.1ijkl

0.6±0.0ijkl

1.29 ± 0.02a

0.02±0. 00k

82.9±1. 1b

k

Cont rol 62.6±0. 3.7±0.1hij 5def Mp- 55.6±0. 3.8±0.0ghi Sc 5hij 50.7±0. Td-Sc 3.8±0.0ghi 1lm 59.8±0. Lt-Sp 4.2±0.0def 6fg Mp- 45.7±0. 3.3±0.1kl Sp 5no 54.0±0. Td-Sp 3.8±0.1ghi 4jk (+)-Catechin 64.1±2. Lt-Sc 4.0±0.5efgh 1de Mp- 54.0±0. 3.6±0.0ijk Sc 5jk 51.7±1. Td-Sc 3.4±0.1jkl 2kl 57.6±0. Lt-Sp 3.9±0.0efghi 5ghi Mp- 44.4±0. 3.1±0.1l Sp 4o 55.0±0. Td-Sp 3.9±0.1fghi 9ij ProcyanidinB 2 63.1±0. Lt-Sc 3.8±0.0ghi 5de Mp- 57.8±1. 3.8±0.0ghi Sc 2gh 57.4±1. Td-Sc 3.8±0.0hij 3ghi 61.6±1. Lt-Sp 4.2±0.0defg 3ef Mp- 48.1±1. 3.3±0.0kl Sp 2mn 57.5±0. Td-Sp 3.6±0.0ijk 4ghi ProcyanidinC 1 71.8±1. Lt-Sc 4.4±0.0cd 0bc Lt-Sc

k

0.12±0.01 bc

0.08±0.01 ef

0.10±0.00 d

0.12±0.00 b

0.00±0.00 k

0.03±0.00j

28

62.0±0. 6ef 64.9±0. Td-Sc 7d 74.1±0. Lt-Sp 7b Mp- 51.2±0. Sp 3l 69.7±0. Td-Sp 2c MpSc

4.3±0.0def

4.7±0.0kl

0.6±0.0ijkl

4.9±0.0b

4.9±0.1hijk

0.6±0.0efghi

4.3±0.0de

3.9±0.0n

0.5±0.0m

3.8±0.0ghi

5.5±0.0cde

0.7±0.0bcd

4.7±0.0bc

5.1±0.1fgh

0.6±0.0bcde

0.45 ± 0.01j 1.05 ± 0.01c 0.97 ± 0.01d 0.51 ± 0.00j 0.77 ± 0.00i

0.11±0.00 c

0.09±0.00 de

0.00±0.00 k

0.04±0.00i

0.00±0.00 k

0.00±0. 00k 0.00±0. 00k 0.00±0. 00k 0.04±0. 01k 0.01±0. 00k

72.1±0. 6efg 76.5±0. 7c 83.8±0. 7b 62.0±0. 3mn 81.0±0. 3b

Different letters in the same column indicate significant differences between means (p<0.05). 1

Comprise the following pigments: delphinidin-3-glucoside, cyaniding-3-glucoside, petunidin-3-glucoside, peonidin-3glucoside, malvidin-3-glucoside. 2Comprise the following pigments: delphinidin-3-(6''acetylglucoside), cyanidin-3-(6''acetylglucoside), petunidin-3-(6''-acetylglucoside), peonidin-3-(6''-acetylglucoside), malvidin-3-(6''-acetylglucoside). 3 Comprise the following pigments: cyanidin-3-(6''-pcoumaroylglucoside), malvidin-3-(6''-pcoumaroylglucoside)cis, petunidin-3-(6''-pcoumaroylglucoside), malvidin-3-(6''-pcoumaroylglucoside)trans. 4Comprise the following pigment: malvidin-3-(6''-caffeoylglucoside). 5Comprise the following pigments: malvidin-3-glucoside acetaldehyde (Vitisin B), malvidin-3-glucoside pyruvate (Vitisin A), malvidin-3-(6''-acetylglucoside) pyruvate, malvidin-3-(6''-acetylglucoside) acetaldehyde. 6Comprise the following pigments: malvidin-3-glucoside-4-vinylphenol, malvidin-3-glucoside-4vinylguaiacol. 7Comprise the following pigment: malvidin-3-glucoside-ethyl-catechin.

29

Table3. Colour and total polyphenol index (TPI) assessment in finished wines with UV-Vis spectrophotometry. Mean ± STD (n=3).

Yeast \ Treatment

Control

(+)-Catechin

ProcyanidinB2

ProcyanidinC1

0.11±0.0b 0.11±0.0b 0.10±0.0b 0.14±0.0ab 0.15±0.0a 0.16±0.0a

0.18±0.0a 0.17±0.0a 0.17±0.0a 0.21±0.0a 0.20±0.0a 0.17±0.0a

0.28±0.0a 0.27±0.1a 0.26±0.1a 0.27±0.0a 0.25±0.0a 0.28±0.0a

0.15±0.0a 0.16±0.0a 0.17±0.0a 0.17±0.0a 0.16±0.0a 0.17±0.0a

1.0±0.0c 1.0±0.0bc 1.1±0.0bc 1.2±0.1b 1.4±0.0a 1.2±0.2b

0.8±0.0c 0.9±0.0bc 0.9±0.0bc 1.0±0.0bc 1.2±0.0a 1.1±0.2b

0.8±0.0d 0.9±0.0bc 0.9±0.0cd 1.0±0.1bc 1.2±0.0a 1.0±0.0b

0.8±0.0d 0.9±0.0c 0.8±0.0d 0.8±0.0cd 1.2±0.0a 1.0±0.0b

4.0±0.7a 4.7±0.7a 3.2±0.7a 3.4±0.4a 4.6±0.9a 3.4±0.2a

12.4±1.4ab 13.5±1.1ab 14.4±0.6a 9.5±0.6b 13.5±1.4a 10.0±0.1b

23.6±0.1b 25.4±0.6a 24.6±0.6ab 23.8±0.5b 25.4±0.5a 24.7±0.5ab

9.4±1.0ab 8.9±0.9ab 7.0±0.8b 11.2±1.2a 10.4±0.9a 7.2±1.0b

A Colour Intensity1 Lt-Sc Mp-Sc Td-Sc Lt-Sp Mp-Sp Td-Sp

B Hue2 Lt-Sc Mp-Sc Td-Sc Lt-Sp Mp-Sp Td-Sp

C Total polyphenol index Lt-Sc Mp-Sc Td-Sc Lt-Sp Mp-Sp Td-Sp 1

Σ of absorption at λmax420nm. λmax 520nm and λmax 620nm

2

Ratio λmax420nm / λmax520nm

Different letters in the same column indicate significant differences between means (p<0.05). Parameters (A, B and C) were treated separately.

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Highlights  (+)-Catechin is prone to forming more polymeric pigments followed by ProcyanidinB2.  Certain non-Saccharomyces yeasts increase pigments in the presence of these flavanols.  Sequential fermentations with Sp(IFI938) yeast increase the amount of polymeric pigments.  Acetaldehyde was found to be key in polymeric pigments condensation.  Non-Saccharomyces help to improve aroma complexity by lowering higher alcohols.

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