Repositorio Institucional

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Selection and characterization of a Patagonian Pichia kudriavzevii for wine deacidification

S.M. del Monaco1, N.B. Barda2, N.C. Rubio3 and A.C. Caballero1,4

1 Grupo de Enologıa, Instituto de Investigacion y Desarrollo en Ingenierıa de Procesos, Biotecnologı´a y Energı´as Alternativas (PROBIEN - Consejo Nacional de Investigaciones Cientı´ficas y Te´ cnicas, CONICET), Facultad de Ingenierıa, Universidad Nacional del Comahue (UNCo), Neuquen, Argentina

2 INTI sede Villa Regina, Villa Regina, Argentina 3 Laboratorio de Toxicologıa y Quımica Legal, Cipolletti, Argentina 4 Facultad de Ciencias y Tecnologıa de los Alimentos, Universidad Nacional del Comahue (UNCo), Villa Regina, Argentina



Keywords indigenous Saccharomyces cerevisiae, malic acid, nonSaccharomyces diversity, wine aroma, wine yeast.

Correspondence Silvana Marıa del Monaco, Grupo de Enologıa, Instituto Multidisciplinario de Investigacion y Desarrollo de la Patagonia Norte (IDEPACONICET), Facultad de Ingenierıa, Universidad Nacional del Comahue (UNCo), Buenos Aires 1400 (8300) Neuquen, Provincia del Neuquen, Argentina. E-mails: silmdm@yahoo.com; silvanadelmonaco@gmail.com

2013/2596: received 31 December 2013, revised 7 May 2014 and accepted 16 May 2014

doi:10.1111/jam.12547



Abstract

Aims: The purpose of this study was to select autochthonous yeasts with metabolic ability to degrade L-malic acid for its potential use in young wine deacidification. Methods and Results: Fifty seven Patagonian nonSaccharomyces yeast of oenological origin were identified by conventional molecular methods and tested in their capability to grow at the expense of L-malic acid. Only four isolates belonging to Pichia kudriavzevii species showed this property, and one of them was selected to continue with the study. This isolate, named as P. kudriavzevii N~ NI15, was able to degrade L-malic acid in microvinifications, increasing the pH 0Á2–0Á3 units with a minimal effect on the acid structure of wine. Additionally, this isolate produced low levels of ethanol, important levels of glycerol (10Á41 Æ 0Á48 g lÀ1) and acceptable amounts of acetic acid (0Á86 Æ 0Á13 g lÀ1). In addition, it improved the sensorial attributes of wine increasing its fruity aroma. Conclusions: The selection of yeasts for oenological use among nonSaccharomyces species led to the finding of a yeast strain with novel and interesting oenological characteristics which could have significant implications in the production of well-balanced and more physicochemical and microbiological stable young wines. Significance and Impact of the Study: The use of P. kudriavzevii N~ NI15 as mixed starter with S. cerevisiae would eliminate the cultural and cellar operations undertaken to adjust the musts acidity, therefore improving wine quality and reducing production costs.



Introduction

Wine is a highly complex mixture of compounds which largely define its appearance, aroma, flavour and mouthfeel properties. Among these compounds, organic nonvolatile acids have a direct impact on quality of wine and imbalances in this fraction can affect its physicochemical and sensory properties, mainly mouth-feel (Beelman and Gallander 1979; Ruffner 1982; Henick-Kling 1993; Radler



1993; Gao and Fleet 1995; Gawel et al. 2007), as well as altering its microbiological estabilty (Delcourt et al. 1995; Pretorius 2000).

L-tartaric and L(-)malic acids are the most important constituents of organic nonvolatile acid fraction in grapes and grape musts, accounting for 90% of the titratable acidity, followed by minor concentrations of citric and lactic acid. Succinic and keto acids are present only in trace amounts in grapes, but their concentration



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is higher in wines as a result of the fermentative metabolism of micro-organisms, mainly yeasts, associated with winemaking (Whiting 1976; Fowles 1992; Radler 1993; Swiegers et al. 2005). Several factors such as grapevine variety, vineyard agricultural practice, temperature, humidity and berry maturity degree, among others, may affect organic nonvolatile acid concentration in grape musts (Ruffner 1982; Flanzy 2000; Volschenk et al. 2001). In particular, L(-)malic acid content, directly related to respiratory quotient of berries, is higher in grape musts from cooler regions than those from warmer regions (Ribereau-Gayon et al. 2006). In the Comahue region, located in the Argentinean North Patagonia, which is one of the southernmost winegrowing regions of the world, malic acid concentrations account for 56% of red grape must titratable acidity, reaching 66% in Pinot noir (Caballero et al. 2005), the emblematic regional vine variety (Weizman 2009). Additionally to its contribution to wine acidity, malic acid represents a fermentable substrate for other micro-organisms which can spoil the wine before and after bottling (du Toit and Pretorius 2000). Without adjustment of acidity, the wines will be regarded as unbalanced or spoilt (Swiegers et al. 2005) hence, malic acid final concentration in wine is of great concern for winemakers and researchers.

The ‘wine yeast’ Saccharomyces cerevisiae does not degrade efficiently malic acid because of the absence of a malate permease (Van Vuuren et al. 1995) and the high Km value of its malic enzyme for this substrate (Fuck et al. 1973; Kuczynski and Radler 1982; Boles et al. 1998). As a consequence, wine L-malic acid has been historically metabolized through malolactic fermentation (MLF), that is the conversion of L-malic to L-lactic acid and carbon dioxide performed by lactic acid bacteria (LAB; Lonvaud-Funel 1999; Mun~oz et al. 2005). However, spontaneous MLF is a very difficult and unpredictable process in winemaking (Wibowo et al. 1985; Thornton and Rodriguez 1996), and the use of commercial starters to induce and guide the process is not always effective (Coucheney et al. 2005). Thus, nonSaccharomyces yeast species belonging to Schizosaccharomyces (Viljoen et al. 1994, 1999; Thornton and Rodrıguez 1996), Zygosaccharomyces (Baranowski and Radler 1984) and Pichia (Issatchenkia) genera (Okuma et al. 1986; Clemente-Jimenez et al. 2004; Seo et al. 2007; Hong et al. 2010) or engineered S. cerevisiae strains coexpressing yeast malate permease together with either yeast (Volschenk et al. 1997, 2001) or LAB malic enzyme genes (Ansanay et al. 1993; Bauer et al. 2005; Husnik et al. 2006), have been investigated as alternatives to MLF for malic acid degradation during winemaking. Unlike natural strains, engineered ‘wine yeast’ strains are able to degrade all malic



acid present in musts without off flavour production (Pretorius and Høj 2005), yet their use in industrial winemaking has so far been delayed because of consumer anti-GMO aversion (Swiegers et al. 2005).

In this work, Patagonian indigenous nonSaccharomyces yeasts of oenological origin were screened in their capabilities to degradate L(-)malic acid as sole carbon source. Four isolates, identified as Pichia kudriavzevii (formerly Issatchenkia orientalis), were positive for this test and one of them proved its potential to be used in winemaking.

Materials and methods

Yeasts

Wild yeasts were obtained from North Patagonian spontaneous red winemaking carried out either at industrial scale (10 000 l) in regional cellars named as C, F, N, N~ and S (Table 1) or at pilot scale (200 l) in an experimental cellar of the Instituto Nacional de Tecnologıa Agropecuaria, Estacion Experimental Agropecuaria (INTA EEA) Alto Valle (noted as I, Table 1), during 2005–2008 vintages. Malbec, Merlot or Pinot noir Samples from initial (12–14 Baume), middle (six Baume) and end (<1 Baume) fermenting musts were appropriately diluted (10À3–10À7) and aliquots of these were spread onto YEPD agar (g lÀ1: yeast extract 10, glucose 20, peptone 20 and agar 20, pH 4Á5) supplemented with 100 ppm of ampiciline (Sigma, Steinheim, Germany). Plates were incubated at 28°C for 2–3 days and isolated colonies were sticked from plates containing between 30 and 300 colony-forming units (CFU) according to their macroscopic features and frequencies to be re-isolated on agar YEPD. Yeast isolates were preserved on YEPD-agar slants, stored at 4°C and subcultured every 2 months. The cultures were also kept at À20°C with 20% v/v glycerol as a cryoprotectant agent.

Yeast identification

Wild yeast identification was performed by conventional methods (Kurtzman and Fell 1998) and by PCR-RFLP analysis of the ITS1-5Á8S-ITS2 region from the nuclear rDNA gene complex (Esteve-Zarzoso et al. 1999). Gene region amplifications were carried out in a Progene thermocycler (Techne, Cambridge, UK) using ITS1 (50-TCC GTAGGTGAACCTGCGG-30) and ITS4 (50-TCCTCCGC TTATTGATATGC-30) primers already described (White et al. 1990). PCR conditions were as indicated by EsteveZarzoso et al. (1999). Amplified DNAs (0.5–10 lg) were digested without further purification with CfoI, HaeIII and HinfI restriction endonucleases (Roche Molecular Biochemicals, Mannheim, Germany) according to the



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Table 1 5Á8S ITS PCR/RFLP patterns of nonSaccharomyces yeasts associated with spontaneous alcoholic fermentations from Patagonian wines



Restriction fragments (bp)



Source



Species



Amp† Cfo I



Hae III



Hinf I



Isolates number



Fermentation stage



Must type



Aureobasidium pullulans(1)



600



Candida stellata(2)



475



Clavispora lusitaniae‡



380



Dekkera anomala(3)



800



Hanseniaspora uvarum/ Kloeckera apiculata(1) 750



Pichia kudriavzevii/ Candida krusei‡



500



Rhodotorula mucilaginosa‡



600



Torulaspora delbrueckii/ Candida colliculosa(3) 800



190 + 180 + 100 215 + 110 + 80 + 60 210 + 80 + 80 340 + 340 + 120 320 + 310 + 105

210 + 180 + 70 + 50



450 + 150 290 + 180 + 130



2



475



235 + 235



10



2



370



190 + 190



5



1*



800



360 + 190 + 160 + 80



1



750



350 + 200 + 180



1



3



1



9



4



4



8



400 + 100 220 + 170 + 150



1*



1



2



300 + 230



400 + 120 350 + 220



1*



330 + 220 + 150 + 100 800



410 + 380



1



Initial Initial Middle Initial Middle Initial Initial Initial Initial Initial Initial Initial Initial Initial Initial Initial and end Initial Initial



Malbec Merlot

Malbec

Merlot Merlot Merlot Merlot Malbec Merlot Pinot noir Pinot noir Malbec Pinot noir Pinot noir

Merlot Malbec



(1)Sabate et al. 2002; (2)Hierro et al. 2006; (3)Esteve-Zarzoso et al.1999; *identity confirmed by sequencing of 26S rRNAgene D1/D2 domains; †Amplicon (bp); ‡this work.



Cellar and vintage

N~ 2006 N~ 2005

N~ 2006

N~ 2005 F 2005 S 2005 N 2005 N~ 2006 C 2007 C 2008 I 2009 N~ 2006 C 2008 I 2009

N~ 2005 N~ 2005



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supplier’s instructions. PCR products and their restriction fragments were separated on 1Á5% (w/v) and 3% (w/v) agarose gels, respectively, in TAE buffer (45 mmol lÀ1 Tris-borate, 1 mmol lÀ1 EDTA, pH 8). Gels were stained with ethidium bromide (5 lg mlÀ1) and visualized under UV light. A 100-bp DNA ladder marker (Gibco BRL, Gaithersburg, MD) served as size standard.

Additionally, the D1/D2 domains of the 26S rRNA gene of the selected isolated were sequenced using NL-1 (50-GCATATCAATAAGCGGAGGAAAAG-30) and NL-4 (50-GGTCCGTGTTTCAAGACGG-30) primers (Kurtzman and Robnett 1998). Amplified fragments were then purified using the Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany) and sequenced. Sequences of the D1/D2 26S rRNA genes were edited and assembled using MEGA ver. 3.1 software and then subjected to a GenBank BLASTN search to retrieve sequences of closely related taxa.

Malic acid assays

Screening. The ability of yeast isolates to use extracellular L-malic acid as carbon and energy source was assayed using MI broth (g lÀ1: yeast nitrogen base with amino acids 1Á7, (NH4)2SO4 5, L-malic acid 20 and bromocresol green 0Á1, pH: 3Á3; Osothsilp and Subden 1986 slightly modified). Bacteriological tubes containing 5 ml of this medium were inoculated with yeast young culture at a final density of 105 cells mlÀ1 and incubated at 25°C under aerobic (shaking at 120 rev minÀ1) conditions. The presence of malic acid degrading yeasts was visualized by a colour change of green to blue in the medium. Assays using MGI broth (MI broth plus glucose 20 g lÀ1, pH 3Á3) and GI broth (g lÀ1: yeast nitrogen base with amino acid 1Á7, (NH4)2SO4 5, D-glucose 20 and bromocresol green 0Á1, pH 3Á3) and carried out under the same conditions were used as controls.

In all cases, assays using S. cerevisiae N~ IF8, an indigenous yeast strain belonging to a Patagonian cellar, were performed as a comparison.



method. For this purpose, aliquots or appropriate dilutions of culture samples were plated on YEPD plates, incubated at 25°C for 24–48 h and colonies counted. At the end of the assays, yeasts were racked and media pH and composition were analyzed.

In all cases, assays using S. cerevisiae N~ IF8 were performed as comparison.

Microvinification

Chemically defined grape juice with similar nitrogen and acidic fraction composition to Patagonian Pinot noir juice (g lÀ1: glucose 100, fructose 100, potasium tartrate 5, L-malic acid 3, citric acid 0Á2, easily assimilable nitrogen 0.208; pH: 3.5; Henschke and Jiranek 1993, modified) was used for microvinification studies with indigenous P. kudriavzevii and S. cerevisiae N~ IF8. Each yeast strain was plated on YEPD-agar plates and a single colony was picked up, inoculated in 50 ml of YEPD broth (g lÀ1: yeast extract 10, peptona 20, D(+) glucose 20) and incubated at 25°C for 2 days with agitation (160 rev minÀ1 in a Rolco shaker). Afterwards, yeast cells were collected by centrifugation at 8000 g for 10 min at 4°C using a Sorvall RC 5C centrifuge. Yeast pellets were washed twice with cold sterile water and resuspended in 5 ml of each must to be counted in a Neubauer chamber.

The fermentations were carried out at laboratory scale, in 250 ml Erlenmeyers containing 200 ml of sterilized synthetic must and inoculated at a final concentration of 106 cell mlÀ1. They were plugged with glass fermentation traps containing sulphuric acid to allow only CO2 to evolve from the system. Fermentations were carried out at 25°C and their evolutions were determined by weighting. Yeast growth was evaluated by monitoring viable yeast count, determined by plating on YEPD medium using the successive dilution method. Plates were incubated at 28°C for 48 h. On plate, yeast colonies belonging to P. kudriavzevii and S. cerevisiae were easily distinguished from each other for their significantly different macroscopic features. All fermentations were carried out in triplicate.



Growth on malic acid and glucose-malic acid broths. Young

cultures of Pichia kudriavzevii yeast strain grown on

YEPD were inoculated in 200 ml YNB-malic acid broth MB: (g lÀ1: yeast nitrogen base 17, L- malic acid 20) or YNB- glucose-malic acid broth MGB: (g lÀ1: yeast nitro-

gen base 17, glucose 20, L- malic acid 20). Control

assays using glucose as single carbon source were also carried out, GB: (20 g lÀ1). Cultures were maintained at 25°C under aerobic (shaking at 150 rev minÀ1) and

anaerobic conditions, sampled routinely and yeast

growth was analyzed using the viable cell counting



Chemical analysis

Organic acid content in the microvinifications’ culture media was analyzed by HPLC in a Shimadzu LC-9A liquid chromatograph (Shimadzu, Kyoto, Japan), equipped with a C-18 column and UV detection. A solution of K2HPO4 0Á2 mol lÀ1, pH 2Á5 was used as mobile phase with a flux of 0Á7 ml minÀ1. The oven was programmed at 40°C for 20 min. Ethanol was determined by headspace gas chromatography in an Agilent 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA),



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equipped with a flame ionization detector (FID) and a DB-Alc2 capillary column (30 m 9 1Á20 lm). Samples were incubated at 40°C and 250 rev minÀ1 for 5 min, and 250 ll of the headspace was injected (syringe temperature 40°C, split injection mode) using an automatic injector CombiPal Agilent G6500. Nitrogen was used as a carrier with a 1Á6 ml minÀ1 flow rate and terbutanol was used as an internal standard. The injector temperature was 250°C and column temperature was 40°C for 4 min, then increased to 120°C in a 20°C minÀ1 rate ramp.

Glucose, fructose, L(-)malic acid, L(-)lactic acid and glycerol content were assessed by enzymatic detection kits (Megazyme International Ireland Ltd., Bray Business Park, Bray,Co. Wicklow, Ireland), and pH was measured with a pH 510 Benchtop meter (OAKTON Instruments, Vernon Hills, IL).

Titratable and volatile acidity (TitA and VA) were determined according to published standard methods (Amerine and Ough 1980). Briefly, for TitA determination, 10 ml of the juice or fermented product were added in 90 ml of distilled water, and an alcoholic solution of phenolphthalein was added. The solution was titrated with a 0Á1023 N sodium hydroxide solution. VA was removed from the samples by boiling and collected by steam distillation, followed by a titration with NaOH.

The volatile components of the fermented samples were analyzed by the Special Analytical Standards service of the National Wine Institute (Mendoza, Argentina)



according to the following protocol: 100 ml of wine were added with 20 ll of R-2-octanol as an internal standard, and volatiles were extracted by means of solid–liquid extraction using an Amberlite XAD-2 polymeric adsorbent and anazeotropic mixture of pentane-dichloromethane solvents (2 : 1). An essential oil drop of the organic fraction was obtained from a Kuderna-Danish concentrator. The profile of the volatile fraction was analysed by injecting 1 ll of the concentrate (split injection mode) in an HP-6890 gas chromatograph (HewlettÀ Packard, Wilmington, DE), equipped with a FID and a HP-INNOWax capillary column (50 m 9 0Á22 mm i.d., 0Á25-mm-film thickness). Nitrogen was used as a carrier with a 30 ml minÀ1 flow rate with a column head pressure of 15 psi. The injector and detector temperature were 310 and 350°C, respectively, and the air flow rate was 400 ml minÀ1. The oven was programmed at 45°C for 5 min, then increased to 165°C in a 2°C minÀ1 rate first ramp, to 280°C in a 10°C minÀ1 rate second ramp and finally kept constant for 10 min. Results were expressed in mg lÀ1.

Sensorial analysis

Sensorial evaluation of the synthetic wine was performed at 20 Æ 2°C by a panel of six judges from INTI (Instituto Nacional de Tecnologıa Industrial) according to IRAM normatives 20005, 20006 and 20012. Aroma intensity was evaluated using a category scale of five



Malic acid (gl–1)



pH Yeast growth (log cfu ml–1)



(a) 7 6 5 4 3 2 1 0 GI/MGI/MI 1



GI MGI MI 2



GI MGI MI 3



(b) 9 8·5 8 7·5 7 6·5 6 5·5 5 4·5 4 0



25 20 15 10 5 0 24 48 72 96 120 144 168 192 Time (h)



Figure 1 (a) Malic acid degrading yeast screening test. Upper pannel shows pH values for media at 240 h. GI: glucose medium. MGI: glucose + malic acid medium. MI: malic acid medium. (1) Initial condition. (2) Pichia kudriavzevii N~NI15. (3) Saccharomyces cerevisiae N~IF8. Lower pannel: culture media aspect in each condition previously described. (b) Yeast growth (log CFU mlÀ1) for P. kudriavzevii (white symbols)

and S. cerevisiae (black symbols) in culture media supplemented with malic acid (MB, squares) and glucose (GB, triangles) as carbon sources.

Remaining malic acid is also represented (circles).



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Table 2 Physiological and biochemical characteristics of Patagonian Pichia kudriavzevii isolates



Patagonian P. kudriavzevii for winemaking 456



Assimilation tests



Fermentation test



Other tests



Glucose +



Sucrose À



D-mannitol À



Sorbitol* À



D-glucosamine +



Succinic acid +



Citric acid +



DL-lactic acid +



DL and L- malic acid +



Glucose +



Fructose +



L-malic acid +



Growth at 37°C +



Growth at 40°C +



Growth in vitaminfree medium +



L-lysine assimilation +



Cycloheximide resistance (10 mg lÀ1) À



Protease activity† +



*D-glucitol. †Either casein or bovine serum albumin tests.



S.M. del Monaco et al.

points (0 = none, 5 = extreme) anchored at different points with the corresponding references. Samples were evaluated monadically at random order and judges were instructed to rinse with water extensively and thoroughly between samples. A 10 min rest between samples was recommended to avoid fatigue. Each wine was judged in duplicate.

Statistical analysis

Data were expressed as mean values Æ SD (n = number cases). ANOVA for multiple data comparison and Tukey honest significant difference (HSD) post hoc tests (a = 0Á05) were performed for mean comparisons. Data normality and variance homogeneity of the residuals were verified by Lilliefors and Bartlett tests, respectively.

Results

Yeasts identification and malic acid screening

Fifty seven wild wine yeasts isolated from eight Patagonian spontaneous red must fermentations and belonging to eight nonSaccharomyces species (Table 1) were screened for their ability to use L-malic acid as a sole carbon source. For comparative purposes, an indigenous Patagonian, Saccharomyces cerevisiae strain with appropriate oenological behaviour (named N~ IF8), was also evaluated.

Of all yeasts assayed, only four isolates named N~ NI15, CNI308, INI3 and INI9 were positive for L-malic acid test, as shown in Fig. 1 for N~ NI15, and they were initially identified as presumably belonging to Pichia kudriavzevii (ex Issatchenkia orientalis)/Candida krusei species based on molecular results (Table 1) along with results from conventional methods (Table 2). The ITS PCR/RFLP restriction pattern observed for this species was similar than that reported by Granchi et al. 1999; ClementeJimenez et al. 2004 and Hierro et al. 2006; although some minor differences in band size for the amplified product and for the digested fragments were evidenced. However, all isolates showed the vegetative cell morphology (data not shown) and the biochemical behaviour (Table 2) consistent with those described by Kurtzman and Fell (1998) for P. kudriavzevii/C.krusei. As a whole, all isolates showed acidophilic character, temperature tolerance and cicloheximide sensitivity (Table 2). To our knowledge, this is the first report on cicloheximide (actidione) sensitivity for P. kudriavzevii. Sequencing of N~ NI15 D1/D2 26S rDNA domains and the ability to sporulate (data not shown) confirmed its identity as P. kudriavzevii (Kurtzmann et al. 2008). This strain was then selected to continue with the study.

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Patagonian P. kudriavzevii for winemaking



Yeast growth (log cfu ml–1)



Extracellular malic acid (g l–1) pH



(A) 25 a

20 cc

15

10



b



b



b



b b



c



5



0 Initial

conditions



Figure 2 Malic acid broth assays under anaerobic conditions. (A) Remaining extracellular malic acid content (g lÀ1, grey bars) and yeast growth (log CFU mlÀ1, white and black bars) for Pichia kudriavzevii (1) and Saccharomyces cerevisiae (2) cultured in different broths during 7 days. MB: malic acid medium, MGB: glucose + malic acid medium and M + MB: pre-incubation of P. kiudriavzevii to 2% L-malic acid during 170 h previous to MB broth assay. (B) pH values obtained from the final broths. Columns displaying different letters within each assay represent significant diferences (ANOVA and Tukey HSD test n = 2, P < 0Á05).



(B) 3·5



3·0



c



2·5



2·0



1·5



1·0



0·5



0·0 Initial

conditions



MB MGB M+MB 1



a



b



a



MB MGB M+MB 1



8 a



a



7



6

cd 5

d 4



3



2



1



0 MB MGB

2



c



c



MB MGB 2



Influence of medium conditions on yeast malic acid utilization and growth

Figure 1b shows yeast growth and substrate consumption during L-malic acid broth assays carried out under aerobic conditions. Only P. kudriavzevii NN~ I15 was able to grow with L-malic acid as a sole carbon source. The maximal population achieved by P. kudriavzevii in this medium was as high as the observed in glucose broth (c.a. 1Á2 9 108 CFU mlÀ1) although its growth rate (evidenced by the slopes of the growth curves) was lower in malic acid (Fig. 1b).

Regarding substrate consumption, P. kudriavzevii degraded approx. 23% of the L-malic acid within the two first days, reaching 62.3% at the seventh day (Fig. 1b).

Factors such as oxygen and glucose availability influenced the nonSaccharomyces yeast behaviour related to L-malic acid consumption. For the same time period but under anaerobic conditions, P. kudriavzevii showed a



lower biomass increase (one logarithmic cycle) and lower L-malic acid utilization (26%; Fig. 2A 1) than those observed in L-malic broth assays under aerobic incubation (three logarithmic cycles and 62.3%, respectively; Fig. 1b). On the other hand, the ability of P. kudriavzevii to grow in L-malic acid as single carbon source under anaerobic conditions was similar to that observed in glucose broth (Table 3). Nevertheless, its metabolic behaviour was totally different (Table 3). Fermentation of L-malic acid as single carbon source occurred without ethanol production, yielding acetic and lactic acid as main products and increasing pH from 2Á90 Æ 0Á02 to 3Á10 Æ 0Á02. (Table 3 and Fig. 2B 1). The presence of glucose in the assay medium decreased the ability of P. kudriavzevii to degrade L-malic acid but this effect was not significant under anaerobic conditions (Fig. 2A 1). Finally, pre-adapting P. kiudriavzevii to 2% L-malic acid during 170 h previous to MI broth assay significantly increased its ability to consume this substrate (45%;



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Table 3 Biomass production and physico-chemical characteristics of malic acid (MB) and glucose broths (MG) fermented under anaerobic conditions by Pichia kudriavzevii (assay end point: 7 days)



Compound (g lÀ1)



Malic acid broth Unfermented medium



Fermented medium



Glucose broth Unfermented medium



Fermented medium



Glucose L(-)Malic acid Citric acid Fumaric acid Lactic acid Acetic acid Glycerol Ethanol pH Biomass (log CFU mlÀ1)



– 20Á10 Æ 1Á08 – – – – – – 2Á91 Æ 0Á01b 5Á17 Æ 0Á18a



– 14Á69 Æ 1Á25 nd 0Á045 Æ 0Á015 2Á200 Æ 0Á145 4Á500 Æ 0Á255 0Á205 Æ 0Á123 0Á002 Æ 0Á004

3Á10 Æ 0Á02a 6Á52 Æ 0Á61b



20Á00 Æ 1Á40 –

2Á90 Æ 0Á01b 5Á04 Æ 0Á18a



10Á75 Æ 3Á52 nd nd nd 1Á000 Æ 0Á161 0Á700 Æ 0Á145 0Á920 Æ 0Á104 2Á660 Æ 1Á210

2Á87 Æ 0Á02b 6Á37 Æ 0Á28b



Values displaying different superscript letter within the horizontal line are significantly different (ANOVA and Tukey HSD test n = 2, P < 0Á05). nd = nondetected.



Fermentation evolution (g l–1 CO2)



(a)100 9·25

90

8·75 80



8·25 70



60



7·75



50



7·25



40



6·75



30



6·25



20



5·75



10



5·25



0



4·75



0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15



Time (days)



(b) 250



3·90



3·85



200



3·80



3·75

150 3·70



100



3·65



3·60



50



3·55



3·50



0 0



1



2



3



4



5



6



7



8



9 10 11 12 13 14 15 3·45



Time (days)



pH



Yeast growth (log cfu ml–1)



Figure 3 Microvinification analysis in

synthetic must. (a) Fermentation evolution (g lÀ1 CO2, squares) and yeast growth (log CFU mlÀ1, triangles) along 14 days of

fermentation for Pichia kudriavzevii (white

symbols) and Saccharomyces cerevisiae (black

symbols). (b) Total Residual Sugars (TRS; g lÀ1, circles) and pH values (diamonds)

evaluated during the analysis.



TRS (g l–1)



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Table 4 Physicochemical characteristics of wines obtained from synthetic must vinifications carried out by Patagonian Pichia kudriavzevii N~NI15 and Saccharomyces cerevisiae N~IF8 strains at laboratory scale



Wines



Parameters

TRS (g lÀ1)* pH Total acidity (g lÀ1)† Volatile acidity (g lÀ1)‡ Ethanol (GL)§ Glycerol (g lÀ1) Organic acids (g lÀ1)

L (-) Malic acid Citric acid Lactic acid Succinic acid Esters (mg lÀ1) Ethyl acetate Ethyl propanoate Ethyl octanoate Ethyl caproate Higher alcohols (mg lÀ1) Butanol n-Pentanol 2-methylbutanol 3-methylbutanol 1-Phenylethanol



Must

218Á23 Æ 1Á04 3Á55 Æ 0Á01b 5Á85 Æ 0Á68

nd nd nd

3Á01 Æ 0Á28a 0Á35 Æ 0Á21 nd nd

nd nd nd nd

nd nd nd nd nd



P. kudriavzevii

nd 3Á73 Æ 0Á07a 5Á73 Æ 0Á38 0Á86 Æ 0Á13 7Á81 Æ 1Á37b

10Á41 Æ 0Á48a

1Á87 Æ 0Á19c 0Á30 Æ 0Á20 0Á05 Æ 0Á07 0Á30 Æ 0Á14b

nd 29Á85 Æ 2Á30a

0Á24 Æ 0Á07a 0Á71 Æ 0Á10

nd 2Á17 Æ 0Á52 0Á06 Æ 0Á04 0Á07 Æ 0Á03 0Á18 Æ 0Á01



S. cerevisiae

nd 3Á55 Æ 0Á01b 6Á22 Æ 0Á69 0Á60 Æ 0Á06

10Á30 Æ 1Á40a 6Á40 Æ 0Á96b

2Á34 Æ 0Á05b 0Á38 Æ 0Á18 0Á18 Æ 0Á04 0Á60 Æ 0Á01a

8Á9 Æ 2Á1 2Á86 Æ 0.11b 0Á02 Æ 0Á05b nd

4Á08 Æ 1Á15 nd

0Á11 Æ 0Á02 0Á09 Æ 0Á01 nd



Values displaying different superscript letter within the horizontal line are significantly different (ANOVA and Tukey HSD test n = 2, P < 0Á05). nd, nondetected. *Total Reducing Sugars. †Expressed as tartaric acid. ‡Expressed as acetic acid. §Gay Lussac degrees (ml of ethanol in 100 ml of wine).



Fig. 2A 1), and as it happened in the L-acid malic broth assay, medium pH was also significantly increased (Fig. 2B 1). As expected, S. cerevisiae was able to degrade L-malic acid only in the presence of glucose under both aerobic (data no shown) and anaerobic conditions (Fig. 2A 2). However, unlike P. kudriavzevii cultures, media pH were not increased (Fig. 2B 2).

Microvinification

Microvinification studies were carried out using synthetic must as a substrate, with similar nitrogen and acidic fraction composition to Patagonian Pinot noir juice. Cultures with indigenous P. kudriavzevii and S. cerevisiae were performed under anaerobic conditions, emulating wine fermentation. Figure 3 shows the results obtained for both yeast strains. An acceptable yield in biomass was observed in both microvinifications (Fig. 3a). Although both fermentations presented similar sugar concentrations at the end of the process, fermentative efficiency (Fig. 3a) as well as sugar consumption rate (Fig. 3b) was higher



for S. cerevisiae than for P. kudriavzevii. A noteworthy fact, in agreement with what was reported in broth assays, is that P. kudriavzevii was again able to raise significantly the medium pH with a minimal effect on acid structure of the wine (decrease of titratable acidity observed in P. kudriavzevii wine was not significant compared with control, Table 4), whereas in the S. cerevisiae culture pH was constant along the fermentation (Fig 3b).

Analysis of media composition showed a higher ability of P. kudriavzevii to metabolize L-malic acid (38%) compared with S. cerevisiae (22%; Table 4). Although both yeast strains were able to consume all initial hexoses, wine composition also evidenced significant differences in the fermentative behaviour between them (Table 4). Under the assayed conditions, P. kudriavzevii was able to produce important amounts of glycerol but it was a weak producer of ethanol when compared with S. cerevisiae N~ IF8. Both yeasts produced relatively low amounts of succinic acid and relatively high amounts of acetic acid. However, S. cerevisiae produced more succinic acid than P. kudriavzevii, which produced more acetic acid than



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Reduced Alcohol



Global Impact 3·5 3·0 2·5 2·0 1·5 1·0 0·5 0·0



Fruit (*) Pinneapple



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Acetaldehyde

Higher alcohols



Solvent



Cooked pears (*)



Figure 4 Sensory analysis of wines obtained from laboratory scale fermentations of synthetic must inoculated with Pichia kudriavzevii (white circles) or Saccharomyces cerevisiae (black circles). ANOVA and Tukey Test, n = 12. Asterisks indicate statistical diferences (P < 0Á05).



the former (Table 4). Additionally, both yeasts showed similar ability to produce higher alcohols and esters, but significant differences between their particular profiles were observed (Table 4). Pichia kudriavzevii was unable to synthetize ethyl acetate but it showed a good production of ethyl esters from fatty acids when compared with the Saccharomyces yeast (Table 4).

Finally, sensorial analysis evidenced significant differences in aromatic perception between P. kudriavzevii and S. cerevisiae wines. These differences were in favour of the former, which showed a higher fruity and cooked pears aroma than the latter (Fig. 4).

Discussion

Fifty seven indigenous Patagonian yeasts of oenological origin identified as belonging to Hanseniaspora uvarum/ Kloeckera apiculata (53%), Candida stellata (21%), Clavispora lusitaniae (10%), Pichia kudriavzevii (ex Issatchenkia orientalis)/Candida krusei (7%), Dekkera anomala (2%), Rhodotorula mucilaginosa (2%), Torulaspora delbrueckii/ Candida colliculosa (2%) and Aureobasidium pullulans (1%) species (Table 1) were screened in their abilities to degrade L-malic acid as single carbon source. Only four isolates belonging to Pichia kudriavzevii (ex Issatchenkia orientalis)/Candida krusei were positive for this test and one of them, confirmed in its teleomorphic form, was selected to continue with the study. Pichia kudriavzevii is a yeast species often reported in grape musts (Jolly et al. 2006; Fleet 2008) but this is the first report of its presence in Patagonian grape musts. Even though this species was detected in a relatively low frequency it was one of



the few detected in several musts along with H. uvarum, and the only species detected at the final stage of fermentation (Table 1). This result concurrs with what was recently reported for China grape fermentations, where individuals of this species dominated at the end of fermentations (Wang and Liu 2013).

Under the assayed conditions, P. kudriavzevii N~ NI15 displayed an extreme tolerance for high L-malate concentrations, levels reaching up to 22 g lÀ1 of L-malate as a sole carbon source. Either in aerobic or anaerobic conditions, it could degrade the compound partially or totally without any negative effect on cell viability and growth (Figs 1b and 2A and Table 3). Yeast species that are recognized for their ability to metabolize extracellular L-malic acid fall into either the Krebs positive or Krebs negative yeast groups (Volschenk et al. 2003; Saayman and Viljoen-Bloom 2006). Krebs positive species Candida utilis, Candida sphaerica, Hansenula anomala, Kluyveromyces lactis and Kluyveromyces marxianus can consume malic acid and other Krebs cycle intermediates as sole carbon and energy source. Krebs negative species S. cerevisiae, Zygosaccharomyces bailii, Schizosaccharomyces pombe and S. pombe var. malidevorans can consume malic acid only in the presence of glucose or another assimilable carbon source. The capability of P. kudriavzevii N~ NI15 to grow in agar plates with L-malic acid and other Krebs cycle intermediates as a sole carbon source (Table 2) as well as its competence to degrade and grow in L-malic broth assays under aerobic and anaerobic conditions (Figs 1b and 2A, Tables 2 and 3) are consistent with a Krebs positive yeast. Additionally, this ability was induced by the substrate, although, unlike with



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what was reported for Krebs positive yeasts (Saayman and Viljoen-Bloom 2006), it was not repressed in the presence of glucose, effect particularlly notable under anaerobic conditions (Fig. 2A 1). This last property constitutes an advantage for the application of P. kudriavzevii N~ NI15 in winemaking where high amounts of glucose are present during most of the process. On the other hand, Taillandier and Strehaiano (1991) showed that under anaerobic conditions, S. pombe completely metabolized L-malate to ethanol and CO2. In this pathway, referred as the maloethanolic fermentation, Lmalate is decarboxylated to pyruvate by the malic enzyme, with further decarboxylation to acetaldehyde by pyruvate decarboxylase and subsequent reduction to ethanol by alcohol dehydrogenase (Saayman and ViljoenBloom 2006). Results showed in Table 3 evidence the absence of maloethanolic fermentation by P. kudriavzevii N~ NI15 in L-malic broth assays. While maloethanolic fermentation is a dissimilatory pathway, this result is consistent with the capability observed for this yeast to grow in these assays (Fig. 2a and Table 3). Additionally, significant pH increases in all fermented media were observed (Fig. 2B).

Vinifications of synthetic musts carried out at laboratory scale confirmed the behaviour of P. kudriavzevii regarding L-malic acid consumption and the effect observed on pH in broth assays. Pichia kudriavzevii N~ NI15 was able to degrade 36% of L-malic acid from de must, increasing significantly its pH in 0Á2–0Á3 units (Fig. 3b) with minor changes in the acidic structure of wine which is evidenced by the titratable acidity value (Table 4). Similar effects on pH have been observed in raw compost material inoculated with indigenous strains of this species after 2 days cultures under anaerobic conditions (Nakasaki et al. 2013).

Acidity adjustment in grape must is an essential step during vinification. In high-acid/low-pH grape musts, typically found in cool-climate regions (pH below 2Á9), reduction of TA prior to fermentation is a prerequisite as the onset of alcoholic fermentation by strains of Saccharomyces will be negatively affected at such extremely low pH. Viticulturists and winemakers have available several vineyard practices (adequate canopy management, trellising and leafpruning techniques) as well as several cellar operations (skin contact, carbonic maceration, among others) to decrease the acidity of grape musts (Volschenk et al. 2006) with a consequent cost in time and money. Additionally, low pH in final wine can be adjusted by blending or, more routinely, by bacterial malolactic fermentation. Although this step is considered the most natural method for wine acidity adjustment, which also contributes to microbial stability and organoleptic complexity, there are a number of pitfalls associated with this



biological process (Henick-Kling 1993). In this context, the use of P. kudriavzevii N~ NI15 as wine starter would eliminate the cultural and cellar operations undertaken to adjust must acidity, favouring the elaboration of wellbalanced, more physicochemical and microbiological stable wines.

Glycerol and acetic acid are the most important byproducts of hexose fermentation. When wine glycerol concentration is near 5Á2 g lÀ1, it has a slightly sweet taste leaving an impression of smoothness on the palate (Noble and Bursick 1984) whereas acetic acid concentrations higher than 1Á0 g lÀ1 have a negative effect on wine taste and flavour (Swiegers et al. 2005). The capability of P. kudriavzevii N~ NI15 to produce amounts of glycerol that exceeded this threshold and amounts of acetic acid lower to this threshold level (Table 4) can be considered an advantage for its use in oenology. As glycerol production is largely the result of a stress response, particularly osmoregulation, and redox balance (Hohmann 1997; Remize et al. 2003), it is plausible that the amount of glycerol produced by this yeast is related to its response against high osmotic pressure present in musts at initial fermentation stages. In this sense, the use of a P. kudriavzevii–S. cerevisiae mixed starter in a sequential form could be an adequate strategy for the production of wines with improved sensorial properties.

Indeed, the must fermented with P. kudriavzevii N~ NI15 presented a pleasant ‘fruity’ aroma which was significantly higher than the one detected in the S. cerevisiae wine. A correlation study between volatile fermentation products and sensory descriptors has shown that compounds positively associated with fruit attributes include ethyl propanoate, ethyl octanoate, ethyl dodecanoate, phenylethyl acetate, 2-methylbutanol, 3-methylbutanol and phenylethanol, among others (Torrea et al. 2011). The presence of some of these compounds in the P. kudriavzevii synthetic wine in concentrations that exceeded their threshold levels and in higher proportions than what was observed in the S. cerevisiae wine (Table 4) could explain the sensorial differences between wines (Fig. 4).

Pichia kudriavzevii is one of the yeasts species included in the 2002 IDF inventory, an authoritative lists of micro-organisms with a documented use in food and published as a result of a joint project between the International Dairy Federation (IDF) and the European Food and Feed Cultures Association (EFFCA; Bourdichon et al. 2012). Although additional assays using natural grape musts and other fermentation scales must be carried out to confirm the behaviour of P. kudriavzevii, the results present in this work position this yeast as a promissory strain with potential application in mixed starters for the production of well-balanced and more physicochemical and microbiological stable young wines.



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Acknowledgements

This work was supported by grants from Universidad Nacional del Comahue (Proyecto de Investigacion 04 l001 Levaduras y Bacterias Lacticas para la diferenciacion de vinos Patagonicos) and MINCyT (PICT SU 2804/ 12).

Conflict of Interest

No conflict of interest declared.

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