effect of inoculation strategy of non-saccharomyces yeasts on...

Effect of inoculation strategy of non-Saccharomyces yeasts on fermentationcharacteristics and volatile higher alcohols and esters in Campbell

Early wines

S.-B. LEE1, C. BANDA1 and H.-D. PARK1,2

1 School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, South Korea; 2 Institute ofFermentation Biotechnology, Kyungpook National University, Daegu 41566, South Korea

Corresponding author: Dr Heui-Dong Park, email [email protected]

AbstractBackground and Aims: Wine made from the Campbell Early grape cultivar has less flavour than wine made fromEuropean grape cultivars. The aim of this study was to investigate the individual fermentation characteristics of several non-Saccharomyces yeasts for improving the aroma of Campbell Early wine.Methods and Results: Nine species of non-Saccharomyces yeasts were used as wine starters in single or co-fermentationwith Saccharomyces cerevisiae. Several fermentation characteristics and physiological properties were investigated. Volatilehigher alcohol and ester compounds were also analysed by GC/MS and by principal component analysis. Single-fermentedwines showed different fermentation kinetics, whereas co-fermented wines showed similar, but slightly different, fermenta-tion kinetics depending on their ethanol tolerance. Principal component analysis indicated that the composition of bothvolatile higher alcohols and esters was distinguishable between single and co-fermented wines, but volatile esters inco-fermented wines were more widely distributed compared to that in single fermented wines.Conclusions: The fermentation kinetics of each strain was different. Volatile esters were more affected by co-fermentationthan volatile higher alcohols, which were produced during the early phase of fermentation. Moreover, interactions amongthe various volatile aromatic compounds affected the final wine aroma.Significance of the Study: These findings could provide valuable information to researchers and winemakers on theenhancement of wine aroma using non-Saccharomyces yeasts.

Keywords: Campbell Early wine, fermentation characteristic, inoculation type, non-Saccharomyces yeast, volatile aromatic compounds

IntroductionWine has a long history and is made by a complex interactionamong yeasts, lactic acid bacteria and fungi (Heard and Fleet1985, Zagorc et al. 2001). Most Korean winemakers have usedSaccharomyces cerevisiae as a wine starter owing to its high etha-nol productivity and ability to protect wine from unfavourablemicroorganisms during fermentation (Philliskirk and Young1975, Casey and Ingledew 1986).

Campbell Early grape, the predominant cultivar grownin Korea, is not competitive with the European grape culti-vars for wine fermentation because of its high malic acidconcentration caused by early harvesting to enhance grapecolour (Lee et al. 2016a). Moreover, chaptalisation is neces-sary for winemaking since the cultivar has low TSS (14–15�Brix), which results in a wine of low flavour (Hwangand Park 2009). To improve wine quality or to reduce malicacid concentration, several strategies, such as MLF, mixingwith different grape cultivars, carbonic maceration and co-fermentation with non-Saccharomyces yeasts, have beenattempted (Pozo-Bayón et al. 2005, Yook et al. 2007, Changet al. 2011, Sadineni et al. 2012, Hong and Park 2013, Huet al. 2016, Lee et al. 2016b). Among these approaches,inoculation of grape must with non-Saccharomyces yeasts hasbeen widely utilised.

Non-Saccharomyces yeasts have been considered of minorsignificance or as spoilage yeasts for a long time, since some

species such as Hanseniaspora and Zygosaccharomyces producean excessive concentration of acetic acid during fermenta-tion (Loureiro and Malfeito-Ferreira 2003, Padilla et al.2016). Several recent studies, however, have reported thatnon-Saccharomyces yeasts can contribute to an improvementof wine quality by producing aromatic compounds, such asesters, terpenes, acids and higher alcohols, during the earlystage of fermentation (Hu et al. 2016, Varela et al. 2016,Whitener et al. 2017). The population and diversity of non-Saccharomyces yeasts are affected by several factors, such asgrape cultivar, climate conditions, localisation, specificweather conditions in each year (vintage), and ripeningphase, which can intimately influence quality and distinc-tion of local wines (Querol et al. 1990, Schütz and Gafner1993, Jolly et al. 2006, Brilli et al. 2015). In addition, someresearchers have indicated that indigenous yeasts can con-tribute to distinctive local wines, based on the grape cultivar,geographical region and composition of targeted fruits(Heard and Fleet 1985, Querol et al. 1992, Schütz andGafner 1993, Mercado et al. 2007, Hong and Park 2013).

Among the volatile aromatic compounds of wine fer-mented by yeasts, higher alcohols are the largest group andplay an important role as ester precursors (Soles et al. 1982,Lambrechts and Pretorius 2000). They are typically classifiedinto: (i) the aliphatic alcohol group (e.g. propanol, isoamylalcohol, isobutanol and active amyl alcohol) and; (ii) the

doi: 10.1111/ajgw.12405© 2019 Australian Society of Viticulture and Oenology Inc.

384 Effect of non-Saccharomyces yeasts on winemaking Australian Journal of Grape and Wine Research 25, 384–395, 2019

https://orcid.org/0000-0001-5042-036X

mailto:School of Food Science and BiotechnologyKyungpook National UniversityDaegu41566South KoreaInstitute of Fermentation BiotechnologyKyungpook National UniversityDaegu41566South Korea

aromatic alcohol group (e.g. 2-phenylethyl alcohol andtyrosol) (Swiegers et al. 2005). Rapp and Mandery (1986)reported that higher alcohols have both positive and nega-tive effects on wine aroma when the concentration ofhigher alcohols is below 300 mg/L and exceeds 400 mg/L,respectively. Esters, a group of the most plentiful com-pounds in wine, comprise two major groups affecting theflavour of fermented beverages: (i) acetate esters, such asethyl acetate, isoamyl acetate, iso-butyl acetate,2-phenylethyl acetate and hexyl acetate, which are com-posed of acetate and ethanol (or a complicated alcoholderived from amino acid metabolism), and are synthesisedby acetyltransferase (AAT) using alcohol and acetyl-CoAand (ii) ethyl esters, such as ethyl hexanoate, ethyloctanoate and ethyl decanoate, which are composed of etha-nol and a medium-chain fatty acid (MCFA), and are formedby two mechanisms, including esterification catalysed by fattyacid ethyl ester synthases/carboxylesterases and alcoholysiscatalysed by acyl-CoA: ethanol O-acyltransferases (Rojaset al. 2001, Saerens et al. 2006, 2008, Padilla et al. 2016, Huet al. 2018). For the last few decades, the impact of thesecompounds on the composition of wine aroma and theirmechanisms of formation in non-Saccharomyces yeasts havebeen revealed by many researchers. Nevertheless, mostresearchers have focused on co-fermentation with non-Sac-charomyces yeasts and S. cerevisiae, and few have studied theeffect of individual non-Saccharomyces yeasts on fermentationof grape juice. Therefore, understanding the fermentationcharacteristics of each non-Saccharomyces yeast and their effecton final wine aroma are still necessary for improving winequality.

In this study, we investigated the changes in several fer-mentation characteristics and physicochemical properties ofCampbell Early wines fermented by nine species of previouslyisolated indigenous non-Saccharomyces yeasts. Inoculationstrategies, such as fermentation with single non-Saccharomycesyeasts and co-fermentation with non-Saccharomyces yeasts andS. cerevisiae, were investigated for wine fermentation to revealthe distinctive characteristics derived from each species. Vola-tile aromatic compounds of all wines were analysed usingprincipal component analysis (PCA), in addition, to sensoryevaluation of the wines.

Materials and methods

Yeast species and growth mediumSaccharomyces cerevisiae W-3, an industrial wine yeast(Yokotsuka and Matsudo 1992), and nine species of non-Saccharomyces yeasts, namely Wickerhamomyces anomalusJK04, Torulaspora delbrueckii JK08, Starmerella bacillarisMR35, Candida quercitrusa P6, Pichia kluyveri P11, Han-seniaspora vineae S7, Hanseniaspora uvarum S8, Candidarailenensis S18 and Metschnikowia pulcherrima S36, were usedfor wine fermentation. All non-Saccharomyces yeasts used inthe present study were previously isolated from food mate-rials, such as nuruk, Muscat Bailey A grape, persimmon andSémillon grape (Wahyono et al. 2016, Jeong et al. 2017, MrS.B. Lee, pers. comm., 2019). All strains were cultured inyeast peptone dextrose (YPD) broth (yeast extract 10 g/L,peptone 20 g/L and glucose 20 g/L) at 30�C for 24 h prior tothe use. Yeast extract and glucose were supplied by Daejung(Siheung, Korea), and peptone was supplied by BD Biosci-ences (San Jose, CA, USA). Moreover, lysine medium(Thermo Fisher Scientific Oxoid, Basingstoke, England)was used to differentiate between S. cerevisiae and non-

Saccharomyces yeasts, because S. cerevisiae does not survive onthis medium.

Wine fermentationCampbell Early grapes (Vitis labrusca cultivar) (14.0�Brix atharvest), cultivated in Sangju, Korea, were washed, stem-med, crushed and chaptalised to 24�Brix to prepare grapemust for wine fermentation. To inhibit growth ofunfavourable bacteria, 200 mg/L potassium metabisulfite(K2S2O5) was added to the 5 kg grape must before inocula-tion with wine yeast. For single fermentation, the yeast cellscultured overnight in YPD medium at 30�C with shaking(150 rpm) were inoculated into 250 g grape must in 1 LErlenmeyer flasks. The flasks were incubated at 30�C withshaking (150 rpm) for 2 days, until the yeast cells reached~108 CFU/mL. They were then inoculated into 5 kg grapemust in a 20 L fermentation container with a vented lid,and each must was fermented at 20�C without shaking untilfermentation was completed. For fermentation withS. cerevisiae and non-Saccharomyces yeasts, the initial culturewas prepared at a ratio of 1:9. For this, S. cerevisiae and non-Saccharomyces yeast, cultured overnight in YPD medium at30�C with shaking (150 rpm), were inoculated into 25 and225 g grape must in 100 mL and 1 L Erlenmeyer flasks,respectively, The following procedure was followed asdescribed above. Final wines were filter-sterilised and storedat 4�C for further analysis and sensory evaluation.

Analytical methodsAll wine samples were centrifuged (3578 g, 10 min) beforeanalysis. To determine the viable cell count of wines, eachsample was serially diluted with 0.85% NaCl to give approx-imately 30–300 CFU/plate. Each co-fermented wine samplewas then spread onto YPD and lysine agar media plates, andeach single-fermented wine sample was spread onto YPDagar media plates. The YPD and lysine plates were incubatedat 30 and 25�C for 24 h and 5–7 days, respectively. The num-ber of S. cerevisiae was counted by subtracting the colonynumbers of non-Saccharomyces yeasts formed on lysinemedium from the total count obtained on YPD agar medium.

Total soluble solids was measured with a refractometer, inaccordance with AOAC guidelines (Caputi 1995) and reduc-ing sugar concentration was analysed using dinitrosalicylicacid according to AOAC guidelines (Caputi 1995). Alcoholconcentration was measured with a hydrometer based on thespecific gravity of wine distillates [expressed as % (v/v)] at15�C (Caputi 1995). pH was measured with a pH meter(Mettler-Toledo, Schwerzenbach, Switzerland) and TA[expressed as tartaric acid (%)] was determined by titrationof filtrates with 0.1 N NaOH.

The concentration of phenolic substances was determinedwith the Folin–Ciocalteau method (Singleton and Rossi1965). Hue and intensity values were obtained from OD420/OD520 and OD420 + OD520 (Kim et al. 2008). The concentra-tion of free sugars and of organic acids was determined byHPLC (Model Prominence, Shimadzu, Kyoto, Japan) with aSugar-Pak I column (diameter 6.5 × 300 mm; Waters, Mil-ford, MA, USA) and PL Hi-Plex H column (diameter7.7 × 300 mm; Agilent Technologies, Santa Clara, CA, USA).The chromatography conditions for the free sugars were asfollows: flow rate, 0.5 mL/min; temperature, 90�C andmobile phase, 50 mg/L Ca-ethylenediaminetetraacetic acid(Ca-EDTA) buffer (Kim et al. 2018). The chromatographyconditions for the organic acids were as follows: flow rate,0.6 mL/min; temperature, 65�C and mobile phase, 0.005 mol

© 2019 Australian Society of Viticulture and Oenology Inc.

Lee et al. Effect of non-Saccharomyces yeasts on winemaking 385

sulfuric acid. Free sugars and organic acids were detectedwith a refractive index detector (RID-10A, Shimadzu) (Hongand Park 2013).

Analysis of volatile aromatic compoundsThe volatile aromatic compounds were determined with a7890A GC/MS (Agilent Technologies) equipped with aflame ionisation detector (FID) (Lee et al. 2016b). The com-pounds were separated on a DB-WAX column(60 m × 250 μm × 0.25 mm; Waters). The detector was anAgilent Technologies 5975C Inert XL MSD with a Triple-Axis detector. Helium was the carrier gas at a constant flowrate of 1 mL/min. The chromatographic oven was initiallyheld at 40�C for 2 min, increased at 2�C/min to 220�C,increased continuously at 20�C/min to 240�C, and thenmaintained at 240�C for 5 min. Volatile aromatic com-pounds were collected with a solid-phase microextraction(SPME) fibre (50/30 μm DVB/CAR/PDMS; Supelco, Bell-efonte, PA, USA). The volatile aromatic compounds wereextracted from the wine in headspace (HS) mode with mag-netic stirring. The sample (5 mL) was placed in an HS vial(20 mm, PTFE/silicon septum, magnetic cap) and 1.25 g ofNaCl was added to increase the concentration of volatilearomatic compounds in the HS. Prior to extraction, the sam-ple was shaken in a water bath at 35�C for 20 min toachieve equilibrium. Subsequently, the SPME fibre wasspiked into the vial and held at 35�C for 40 min. Volatilearomatic compounds were identified by a comparison oftheir GC retention time and MS with spectral data from theWiley9Nist 0.8 library mass spectral search program, version5.0 (Torrens et al. 2004).

Sensory evaluationA five-point hedonic scale was used for sensory evaluation.Before being subjected to sensory evaluation, each winewas place in a sample bottle and left undisturbed at roomtemperature for 1 h with the bottle lid closed. After flavourevaluation, each wine was poured into wine glasses to eval-uate the colour, sweetness, sourness and overall preference.The panel was composed of 20 judges with sensitive tastediscrimination from the Department of Food Science andTechnology, Kyungpook National University, Korea. Eachjudge evaluated the wines with an at least a 3 min intervalbetween samples and water was provided to cleanse the pal-ate. Sensory scores were assigned as follows: 5 (excellent),3 (fair) and 1 (very poor).

Statistical analysisAll experiments were in triplicate, and results were analysedwith SAS software (version 9.4, SAS Institute, Cary, NC,USA). Significance was determined at a threshold ofP < 0.05 using ANOVA, followed by the Duncan’s multiplerange test (Kim et al. 2008). Similarities were determinedamong different wine samples by PCA, reducing the dimen-sion from variables to two principal components, and keep-ing most of the original information in the data set.Principal component analysis was conducted with SAS soft-ware. In addition, correlation coefficients of volatile com-pounds identified by SPME/GC/MS were analysed byPearson’s correlation analysis (Chung et al. 2016).

Results

Fermentation characteristics of Campbell Early wineFigure 1 shows the TSS, and concentration of reducingsugars and of alcohol of Campbell Early wine fermentedindividually with S. cerevisiae and each of the nine species ofnon-Saccharomyces yeasts isolated previously and co-fermented with each of those strains together withS. cerevisiae. The alcohol fermentation of wine co-fermentedand fermented with S. cerevisiae (control) was completedwithin 7 days, and the alcohol concentration of these winesreached 12.4–13.0%. Wine fermented, however, with eachof the nine species of non-Saccharomyces yeasts showed fer-mentation kinetics that depended on the species. Alcoholfermentation by S. bacillaris occurred as the earliest butslowest among all single-fermented wines with non-Saccha-romyces yeasts (23 days for fermentation). Hanseniasporavineae began to produce alcohol on day 2 of fermentationand completed fermentation on day 11, which was thefastest among all single-fermented wines. Other non-

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Figure 1. Effect of fermentation of Campbell Early grape must with eitherSaccharomyces cerevisiae W-3 and or a single strain of several non-Saccharomyces species ( , , ) and of co-fermentation with S. cerevisiaeW-3 together with one of several non-Saccharomyces species ( , , ) onthe TSS ( , ), and concentration of reducing sugars ( , ), and alcohol( , ). (a) S. cerevisiae W-3; (b) Wickerhamomyces anomalus JK04;(c) Torulaspora delbrueckii JK08; (d) Starmerella bacillaris MR35;(e) Candida quercitrusa P6; (f) Pichia kluyveri P11; (g) Hanseniasporavineae S7; (h) H. uvarum S8; (i) Candida railenensis S18; and(j) Metschnikowia pulcherrima S36.



Saccharomyces yeasts began alcohol fermentation at days7–11 and completed fermentation at days 15–23. The pHand TA of wines fermented with S. cerevisiae, such as theControl and co-fermented wines, rapidly decreased andincreased in the early phase of the fermentation, and finallyreached 3.50–3.58 and 0.51–0.58%, respectively (Figure 2).In contrast, the kinetics of wines fermented with a singlespecies varied depending on the species. The TA of winesfermented with W. anomalus and T. delbrueckii significantlyincreased during the early phase of fermentation andreached a maximum value at the middle phase; the pH ofthese wines decreased throughout the early phase of fer-mentation and slightly increased after the middle phase. TheTA and pH of wine fermented with S. bacillaris sharplyincreased and decreased, respectively, at the beginning offermentation and gradually decreased and increasedthroughout the middle and latter phases. The TA and pH ofwine fermented with H. vineae did not change notably com-pared to that of other wines. Moreover, the TA and pH of

wines fermented by the other individual species steadilyincreased and decreased, respectively, throughout fermenta-tion. Generally, the pH of wines fermented with a singlespecies was higher than that of wines co-fermented,whereas TA of wines fermented with a single species, exceptfor W. anomalus, was lower than that of wines co-fermented.Viable cell counts of wine fermented with H. uvarum andC. railenensis decreased considerably in the early phase offermentation and increased significantly after day 5 of fer-mentation (Figure 3), whereas those of other wines fer-mented with a single species slightly decreased at thebeginning, increased again, and were maintained at>7.5 log CFU/mL. For co-fermented wines, the viable cellcount of non-Saccharomyces yeasts, except for S. bacillaris andH. vineae, rapidly decreased after the middle phase of fer-mentation since alcohol concentration was produced andthose species, except for W. anomalus, completely died whenfermentation was completed. Moreover, that of S. bacillarisMR35 somewhat decreased when the wine alcohol concen-tration reached 12%. Conversely, that of H. vineae remainedat a high level throughout fermentation, implying that thosetwo species have an ethanol tolerance higher than that ofother non-Saccharomyces yeasts. The concentration of pheno-lic substances of wines fermented with a single species ofnon-Saccharomyces yeasts, except for S. bacillaris, increaseduntil the middle phase of fermentation and then decreasedgradually, whereas that of S. bacillaris did not change nota-bly during alcohol fermentation (Figure 4). The concentra-tion of phenolic substances of all co-fermented winesgenerally increased throughout alcohol fermentation.

Concentration of free sugars and organic acids in CampbellEarly wineControl and co-fermented wines consumed glucose andsucrose completely (Table 1). In addition, a low concentra-tion of fructose was detected. In most wines fermented witha single species, some free sugars, such as glucose, sucroseand fructose, were not completely consumed; the concen-tration of fructose of wines fermented with C. quercitrusa,P. kluyveri and H. vineae was higher than that in other wines.The concentration of citric acid of wines fermented witheach of the non-Saccharomyces yeasts, except forW. anomalus, was lower than that of co-fermented wines.Furthermore, the concentration of malic acid of wines fer-mented with W. anomalus and H. uvarum was the lowest,and that of wine fermented by H. vineae was the highestamong all wines. The concentration of succinic acid of allwines fermented with non-Saccharomyces yeasts, except forS. bacillaris, was lower than that of co-fermented wines,whereas the concentration of tartaric acid and lactic acidwas not significantly different between the wines. Aceticacid concentration of wines fermented with individual spe-cies was slightly higher than that of Control and co-fermented wines.

Volatile aromatic compounds of Campbell Early wineEight volatile higher alcohols were detected in all wines(Table 2), with each alcohol showing a variable patterndepending on the species and inoculation strategy. The con-centration of 1-propanol of wines single-fermented withT. delbrueckii, P. kluyveri, H. uvarum and C. railenensis was sig-nificantly higher than that of co-fermented wines. Further-more, the concentration of 1-propanol of some winesfermented by other species was slightly higher than that of

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Figure 2. Effect of fermentation of Campbell Early grape must with eitherSaccharomyces cerevisiae W-3 and or a single species of several non-Saccharomyces yeasts ( , ) and of co-fermentation with S. cerevisiae W-3together with one of several non-Saccharomyces species ( , ) on the pH( , ) and TA ( , ). (a) S. cerevisiae W-3; (b) Wickerhamomycesanomalus JK04; (c) Torulaspora delbrueckii JK08; (d) Starmerella bacillarisMR35; (e) Candida quercitrusa P6; (f) Pichia kluyveri P11;(g) Hanseniaspora vineae S7; (h) H. uvarum S8; (i) Candida railenensis S18;and (j) Metschnikowia pulcherrima S36.



co-fermented wines. Iso-butanol concentration of wines fer-mented by single-culture of S. bacillaris, C. railenensis,M. pulcherrima and T. delbrueckii was significantly higherthan that of co-fermented wines. Iso-amyl alcohol concen-tration of wines fermented by both single- and co-culture ofC. railenensis was the highest among each inoculation type,while those of wines fermented by both single- andco-culture of S. bacillaris were the lowest among each inocu-lation type. The concentration of 1-hexanol in winefermented by non-Saccharomyces yeasts, except forP. kluyveri, was higher than that of co-fermented wines. Fur-thermore, the concentration of 1-heptanol of co-fermentedwines by non-Saccharomyces yeasts, except for C. railenensisand M. pulcherrima, was higher than that of wines fermentedwith individual yeasts. In addition, 1-heptanol concentrationof wines fermented by both single- and co-culture ofH. uvarum and C. railenensis was higher than that of the

control wine. Thus, most non-Saccharomyces yeasts do notproduce 1-heptanol during fermentation. The concentrationof 2-ethyl-1-hexanol of wines fermented by individual non-Saccharomyces yeasts was higher than that of co-fermentedand Control wines, whereas 1-octanol concentration ofwines fermented by individual non-Saccharomyces yeasts,except for H. uvarum, was lower than that of co-fermentedwines. The concentration of 1-nonanol of all wines, exceptfor single-culture of H. vineae, was lower than that of theControl wine. Additionally, 1-nonanol concentration ofsingle-fermented wines by non-Saccharomyces yeasts, exceptfor H. vineae, was lower than that of co-fermented wines.

Principal component analysis of volatile higher alcoholsof all wines indicated that PC1 and PC2 accounted for 51.21and 27.30% of the variance, respectively (Figure 5). ThePCA plot distinguished between two groups, namely single-fermented wines [not inoculated with S. cerevisiae and co-

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Figure 3. Effect of fermentation of Campbell Early grape must with eitherSaccharomyces cerevisiae W-3 and or a single species of several non-Saccharomyces yeasts ( ) and of co-fermentation with S. cerevisiae W-3( ) together with one of several non-Saccharomyces species ( ) on theviable cell count ( , , ). (a) S. cerevisiae W-3; (b) Wickerhamomycesanomalus JK04; (c) Torulaspora delbrueckii JK08; (d) Starmerella bacillarisMR35; (e) Candida quercitrusa P6; (f) Pichia kluyveri P11;(g) Hanseniaspora vineae S7; (h) H. uvarum S8; (i) Candida railenensis S18;and (j) Metschnikowia pulcherrima S36.

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Figure 4. Effect of fermentation of Campbell Early grape must with eitherSaccharomyces cerevisiae W-3 and or a single species of several non-Saccharomyces yeasts ( ) and of co-fermentation with S. cerevisiae W-3together with one of several non-Saccharomyces species ( ) on theconcentration of the phenolic substances. (a) S. cerevisiae W-3;(b) Wickerhamomyces anomalus JK04; (c) Torulaspora delbrueckii JK08;(d) Starmerella bacillaris MR35; (e) Candida quercitrusa P6; (f) Pichiakluyveri P11; (g) Hanseniaspora vineae S7; (h) H. uvarum S8; (i) Candidarailenensis S18; and (j) Metschnikowia pulcherrima S36.



Table1.

Concentra

tionof

freesugars

andorganicacidsin

CampbellE

arlywines

ferm

entedwith

individualnon-Saccharomyces

yeastsandco-fe

rmentedwith


yeaststogether

with

S.cerevisiae.

Freesu

gars

conce

ntration(g/L

)Organ

icac

idco

nce

ntration(g/L

)

Inocu

latedyea

stIn

ocu

lation

strategy

Gluco

seSucrose

Fru

ctose

Citricac

idTartaric

acid

Malic

acid

Succinic

acid

Lac

ticac

idAce

ticac

id

Saccharom

yces

cerevisiae

W3

Single

ND

ND

0.36�

0.01cd

1.05�

0.03efgh

1.84�

0.14a

3.12�

0.11bcd

1.18�

0.08defg

0.19�

0.00d

0.03�

0.01gh

Wickerham

omyces

anom

alusJK

04

Single

0.08�

0.02a

0.04�

0.00a

0.56�

0.02cd

1.33�

0.06c

1.81�

0.13a

2.66�

0.09e

1.04�

0.09fgh

0.22�

0.01bcd

0.45�

0.03a

Co-fermen

ted

ND

ND

0.40�

0.02cd

1.15�

0.04de

2.10�

0.09a

3.33�

0.09b

1.33�

0.10bcde

0.23�

0.0bc

0.03�

0.01gh

Torulaspora

delbrueckiiJK

08

Single

0.06�

0.01ab

0.04�

0.01a

0.66�

0.02cd

0.82�

0.03j

1.75�

0.16a

3.07�

0.10bcd

0.95�

0.08gh

0.24�

0.02b

0.20�

0.02d

Co-fermen

ted

ND

ND

0.72�

0.02c

0.98�

0.02fghi

1.93�

0.12a

2.54�

0.12e

1.42�

0.11bcd

0.23�

0.00bc

0.04�

0.01fgh

Starmerella

bacillarisMR35

Single

ND

ND

0.32�

0.01d

0.90�

0.05ij

1.81�

0.15a

3.16�

0.12bcd

1.33�

0.12bcde

0.21�

0.01bcd

0.29�

0.04b

Co-fermen

ted

ND

ND

0.36�

0.01cd

1.06�

0.05efg

1.95�

0.14a

2.98�

0.07cd

1.09�

0.09efgh

0.20�

0.01cd

0.06�

0.01fg

Can

dida

quercitrusa

P6

Single

0.08�

0.01a

0.01�

0.01b

1.53�

0.13b

0.94�

0.04gh

ij1.68�

0.18a

3.31�

0.14b

0.99�

0.09gh

0.22�

0.01bcd

0.14�

0.02e

Co-fermen

ted

ND

ND

0.45�

0.01cd

1.16�

0.03de

1.99�

0.10a

3.24�

0.09bc

1.28�

0.08cdef

0.22�

0.01bcd

0.02�

0.01gh

Pichia

kluyveriP11

Single

0.07�

0.01a

0.03�

0.00ab

1.49�

0.08b

0.88�

0.06ij

1.78�

0.15a

3.33�

0.09b

0.83�

0.08h

0.19�

0.00d

0.26�

0.03c

Co-fermen

ted

ND

ND

0.38�

0.02cd

1.21�

0.04d

2.03�

0.13a

3.32�

0.13b

1.46�

0.13bc

0.23�

0.02bc

0.02�

0.01gh

Han

seniaspora

vineaeS7

Single

ND

0.03�

0.01ab

7.25�

0.56a

1.10�

0.10ed

f1.84�

0.19a

4.27�

0.17a

1.05�

0.09fgh

0.20�

0.02cd

0.16�

0.02e

Co-fermen

ted

ND

ND

0.40�

0.01cd

1.61�

0.08a

1.93�

0.09a

2.97�

0.06cd

1.27�

0.12cdef

0.20�

0.01cd

0.04�

0.01fgh

H.uvarum

S8

Single

0.07�

0.01a

0.02�

0.01ab

0.36�

0.01cd

0.84�

0.06ij

1.72�

0.14a

2.69�

0.10e

1.11�

0.10efg

0.24�

0.01b

0.16�

0.02e

Co-fermen

ted

ND

ND

0.39�

0.01cd

1.44�

0.10b

2.06�

0.21a

3.33�

0.10b

1.80�

0.14a

0.29�

0.01a

0.04�

0.01fgh

Can

dida

railenensis

S18

Single

0.09�

0.01a

0.03�

0.01ab

0.36�

0.01cd

0.93�

0.07hij

1.76�

0.18a

2.92�

0.12d

1.09�

0.12efgh

0.23�

0.01bc

0.08�

0.01f

Co-fermen

ted

ND

ND

0.37�

0.01cd

1.12�

0.04de

1.94�

0.16a

3.15�

0.08bcd

1.54�

0.11b

0.22�

0.02bcd

0.03�

0.01gh

Metschnikow

iapu

lcherrimaS36

Single

0.04�

0.01b

ND

0.30�

0.01d

0.88�

0.03ij

2.01�

0.20a

3.14�

0.13bcd

0.96�

0.09gh

0.22�

0.01bcd

0.08�

0.01f

Co-fermen

ted

ND

ND

0.37�

0.02cd

1.10�

0.03def

1.83�

0.11a

2.89�

0.11d

1.14�

0.09efg

0.21�

0.01bcd

0.01�

0.01h

Differentletterswithin

thesamecolumnindicateasign

ificantdifference

(P<0.05).ND,notdetected.

Table2.

Concentra

tionof

volatilehigher

alcoholsin

CampbellE

arlywines

ferm

entedwith


yeastsandco-fe

rmentedwith


yeaststogether

with

S.cerevisiae.

Conce

ntrationofvolatile

higher

alco

hols

(mg/L)

Inocu

latedyea

stIn

ocu

lation

strategy

1-Pro

pan

ol

Iso-butanol

Isoam

ylalco

hol

1-Hex

anol

1-Hep

tanol

2-Eth

yl-

1-hex

anol

1-Octan

ol

1-Nonan

ol

Saccharom

yces

cerevisiae

W3

Single

2.20�

0.30e

45.54�

4.35fghi

515.32�

34.90ab

18.56�

1.76gh

2.93�

0.35def

0.58�

0.05f

1.63�

0.14cd

23.89�

2.52ab

Wickerham

omyces

anom

alus

JK04

Single

3.87�

0.30e

38.71�

3.41gh

ij265.01�

28.64g

23.25�

2.15cdefg

1.42�

0.21hi

1.74�

0.15d

0.77�

0.05g

8.40�

0.73j

Co-fermen

ted

2.29�

0.20e

50.86�

5.39efgh

458.08�

40.04bc

18.65�

2.04gh

3.01�

0.27def

0.62�

0.05f

2.68�

0.26a

19.30�

1.62cde

TorulasporadelbrueckiiJK

08

Single

22.84�

2.00c

57.18�

5.22cdef

384.51�

33.77cde

27.89�

2.45bcd

1.05�

0.11i

2.21�

0.19b

0.71�

0.06g

10.18�

0.87ij

Co-fermen

ted

2.26�

0.24e

37.30�

3.38hij

335.61�

30.89efg

17.94�

1.69gh

1.75�

0.21gh

0.53�

0.06f

2.40�

0.18a

16.15�

1.40efg

Starmerella

bacillarisMR35

Single

3.22�

0.39e

103.97�

11.17a

163.06�

13.07h

43.47�

3.98a

0.42�

0.07j

2.85�

0.25a

0.77�

0.06g

7.21�

0.75j

Co-fermen

ted

2.78�

0.22e

56.03�

5.85cdef

273.59�

23.92fg

25.14�

2.83bcdef

1.83�

0.17gh

1.59�

0.14d

1.80�

0.22bc

14.93�

1.53fg

Can

dida

quercitrusa

P6

Single

5.13�

0.42e

34.14�

3.14ij

360.28�

37.82def

22.85�

2.20defg

2.69�

0.32ef

2.41�

0.23b

1.23�

0.10e

9.89�

0.95ij

Co-fermen

ted

2.20�

0.29e

44.88�

4.14fghi

433.95�

41.54bcd

15.96�

1.32h

2.74�

0.25ef

0.58�

0.07f

2.59�

0.27a

18.29�

1.67cdef

Pichia

kluyveriP11

Single

11.07�

0.99d

47.37�

3.53fghi

353.99�

32.39def

17.88�

1.63gh

ND

1.00�

0.10e

0.85�

0.08fg

10.07�

1.04ij

Co-fermen

ted

2.15�

0.19e

53.02�

5.07defg

481.51�

43.54ab

20.47�

1.96fgh

3.53�

0.31d

0.66�

0.05f

2.39�

0.21a

18.73�

1.94cde

Han

seniasporavineaeS7

Single

4.58�

0.49e

26.11�

1.61j

377.81�

32.32cde

27.97�

2.39bcd

1.45�

0.13hi

1.99�

0.19c

1.34�

0.15de

25.11�

2.44a

Co-fermen

ted

2.79�

0.31e

42.85�

4.49fghi

362.08�

35.51def

21.69�

2.09efgh

2.38�

0.21efg

0.54�

0.05f

1.37�

0.15de

21.13�

1.89bcd

H.uvarum

S8

Single

25.75�

0.22b

66.12�

6.15cd

337.89�

30.43efg

29.13�

2.55bc

4.84�

0.42bc

1.61�

0.10d

1.15�

0.13ef

13.74�

1.31gh

Co-fermen

ted

2.74�

0.24e

65.76�

6.51cd

506.08�

42.96ab

20.37�

2.17fgh

5.28�

0.47ab

0.72�

0.07f

0.73�

0.09g

17.53�

1.62def

Can

dida

railenensisS18

Single

46.57�

4.95a

91.36�

7.73b

502.57�

49.26ab

30.72�

2.84b

5.75�

0.49a

1.62�

0.13d

1.29�

0.10e

11.70�

1.25hi

Co-fermen

ted

2.33�

0.23e

69.48�

6.35c

560.82�

51.58a

23.88�

1.92cdefg

4.51�

0.43c

1.05�

0.11e

2.59�

0.12a

21.48�

1.69bc

Metschnikow

iapu

lcherrima

S36

Single

4.45�

0.43e

86.81�

8.48b

481.12�

40.45ab

27.06�

3.42bcde

3.09�

0.35de

1.57�

0.12d

1.11�

0.07ef

11.48�

1.24hi

Co-fermen

ted

1.95�

0.18e

63.82�

5.92cde

473.75�

42.59ab

15.41�

1.72h

2.33�

0.29fg

0.48�

0.04f

1.99�

0.18b

16.44�

1.36efg



ificantdifference




fermented wines, including the Control wine (inoculatedwith S. cerevisiae)], indicating that volatile higher alcohols inCampbell Early wines were influenced by S. cerevisiaebecause the Control wine (single-culture of S. cerevisiae) waslocated in the centre of the other co-fermented wines. Inaddition, single-fermented wines were located on the rightside of each co-fermented wine, depending on the species ofnon-Saccharomyces yeasts.

Sixteen volatile esters were detected in all wines(Table 3), with each ester showing different patternsdepending on the strain and inoculation strategy, similar tovolatile higher alcohols. Methyl acetate concentration ofwines fermented by a single-culture of W. anomalus,P. kluyveri, H. uvarum and C. railenensis was significantlyhigher than that of Control and co-fermented wines. Inaddition, the methyl acetate concentration of single-fermented wines by T. delbrueckii and M. pulcherrima wereslightly higher than that of co-fermented wines. Ethyl ace-tate concentration of wines fermented by a single-culture ofW. anomalus, P. kluyveri, H. vineae, H. uvarum andM. pulcherrima and co-culture of H. vineae and H. uvarumwere significantly higher than that of the Control wine.Isobutyl acetate and isoamyl acetate concentration of single-fermented wines of non-Saccharomyces yeasts was lower thanthat of the Control wine, and that of most co-fermentedwines was higher than that of single-fermented wines(except for S. bacillaris and H. uvarum for isobutyl acetate;P. kluyveri for isoamyl acetate). The concentration of ethylbutyrate, ethyl octanoate, ethyl decanoate, ethyl9-decenoate and ethyl dodecanoate of all single- and co-fermented wines was lower than that of the Control wine.Furthermore, the concentration in most single-fermentedwines was lower than that in co-fermented wines (exceptfor C. railenensis for ethyl butyrate; H. uvarum andC. railenensis for ethyl octanoate; C. railenensis andM. pulcherrima for ethyl decanoate; M. pulcherrima for ethyl9-decenoate; C. railenensis and M. pulcherrima for ethyl dode-canoate). Ethyl hexanoate concentration of most wines fer-mented by single-culture of non-Saccharomyces yeasts wassignificantly lower than that in the Control, and wines fer-mented by single-culture of C. railenensis and M. pulcherrima

showed similar ethyl hexanoate concentration whencompared to that of the Control wine. The concentrationof n-hexyl acetate of wines fermented by single-culture ofnon-Saccharomyces yeasts, except for P. kluyveri, was notablylower than that of the Control wine, whereas ethyl non-anoate concentration of single-fermented wines of non-Sac-charomyces yeasts, except for S. bacillaris and H. vineae, washigher than that of the Control and co-fermented wines.The concentration of methyl salicylate of all wines was notnotably different; however, the concentration in wines fer-mented by single-culture of S. bacillaris and P. kluyveri wasslightly higher than that in the Control wine. Ethyl phen-ylacetate concentration of all wines was mostly similar. Theconcentration of 2-phenylethyl acetate of wines fermentedby single-culture of P. kluyveri and single- and co-culture ofH. vineae was significantly higher; however, that of otherwines was lower compared to the Control wine. Ethylhexadecanoate concentration of co-fermented wines washigher than that in the Control wine, and that of wines fer-mented by single-culture of H. uvarum and C. railenensis wasslightly higher than in the Control. The volatile ester com-pounds of all wines were subjected to PCA, and PC1 andPC2 represented 64.85 and 14.89% of the variance, respec-tively (Figure 6). Similar to the PCA plot of volatile higheralcohol compounds, volatile ester compounds formed twoclusters, such as single-fermented wines (not inoculatedwith S. cerevisiae) and co-fermented wines (inoculated withS. cerevisiae). In addition, single-fermented wines werelocated on the left and upper side of each co-fermentedwine, depending on the species of non-Saccharomyces yeast.

Hue and intensity of Campbell Early wineThe hue and intensity values of all the wines are listed inTable 4. Initial hue and intensity values of wines were1.406 � 0.023 and 3.488 � 0.035, respectively. Hue valuesof all the wines fermented with a single species were higherthan those of the Control and co-fermented wines, whereasthe intensity values of most wines fermented with a singlespecies were lower than those of the Control and co-fermented wines (except for C. quercitrusa). Hue and inten-sity values of wines fermented by W. anomalus, T. delbrueckii,S. bacillaris, H. vineae and M. pulcherrima were, respectively,significantly higher and lower than those of the Control andco-fermented wines.

Sensory evaluationTable 5 shows the sensory evaluation results of CampbellEarly wines fermented by single-culture of non-Saccharomy-ces yeasts and by co-culture of those species and S. cerevisiae.Some wines fermented with a single species that showedrelatively high hue and low intensity values (W. anomalus,T. delbrueckii, S. bacillaris, H. vineae and M. pulcherrima)exhibited lower colour values compared with co-fermentedwines. Furthermore, most wines fermented by a single-culture of non-Saccharomyces yeasts obtained higher scoresfor sweetness (except for C. quercitrusa and C. railenensis) andsourness (except for H. uvarum and M. pulcherrima) com-pared to those of co-fermented wines. As a result of flavourand overall preference, wines fermented by single-culture ofW. anomalus and H. uvarum and co-culture of C. quercitrusaobtained higher scores compared to that of the Controlwine. Additionally, wine fermented by co-culture ofH. uvarum obtained a higher flavour score compared to thatof the Control wine.

Figure 5. Profiling of volatile higher alcohols based on principalcomponent analysis (PCA) and solid-phase microextraction (SPME)/GC/MSanalysis of Campbell Early grape wine fermented by individual species ofnon-Saccharomyces yeasts ( ) and co-fermented by individual species ofnon-Saccharomyces yeasts together with S. cerevisiae W-3 ( ).S. cerevisiae W-3 ( ); Wickerhamomyces anomalus JK04 ( , );Torulaspora delbrueckii JK08 ( , ); Starmerella bacillaris MR35 ( , );Candida quercitrusa P6 ( , ); Pichia kluyveri P11 ( , ); Hanseniasporavineae S7 ( , ); H. uvarum S8 ( , ); Candida railenensis S18 ( , ); andMetschnikowia pulcherrima S36 ( , ).



Table3.

Concentra

tionof

volatileesterc

ompounds

inCampbellE

arlywines

ferm

entedwith


yeastsandco-fe

rmentedwith


yeaststogether

with

S.cerevisiae.

Conce

ntrationofvolatile

esterco

mpoundsin

wine(μg/L)

Inocu

latedyea

stType

Methyl

acetate

Eth

ylac

etate

Isobutyl

acetate

Eth

yl

butyrate

Isoam

ylac

etate

Eth

yl

hex

anoate

n-H

exyl

acetate

Eth

yloctan

oate

Saccharom

yces

cerevisiae

W3

Single

1.05�

0.08fg

306.87�

26.17ef

9.58�

1.04cd

9.74�

1.05a

569.31�

47.47b

174.22�

15.37a

50.19�

5.01b

542.32�

49.28a

Wickerham

omyces

anom

alus

JK04

Single

4.64�

0.40a

1740.35�

131.03a

5.25�

0.46ef

2.63�

0.28h

70.68�

6.85jk

50.59�

4.74e

3.37�

0.22i

121.29�

11.74gh

Co-fermen

ted

1.16�

0.10ef

366.00�

43.8e

11.71�

1.20b

6.76�

0.61cd

486.96�

44.13c

134.06�

14.00b

47.25�

4.28bc

395.51�

40.88c


08

Single

0.96�

0.09fg

289.99�

27.25ef

1.74�

0.20h

3.47�

0.33gh

54.82�

5.79k

45.71�

5.12e

4.91�

0.39i

109.11�

11.71h

Co-fermen

ted

0.74�

0.08g

287.37�

39.89ef

5.51�

0.47e

7.92�

0.70bc

347.88�

36.36e

125.25�

9.57b

42.79�

4.67cd

321.22�

35.84d

Starmerella

bacillarisMR35

Single

ND

138.84�

10.69g

3.57�

0.30fgh

2.52�

0.26h

5.85�

0.56k

10.21�

0.90f

0.51�

0.04i

10.04�

1.07i

Co-fermen

ted

0.49�

0.02h

193.67�

19.72fg

1.76�

0.13h

5.82�

0.67def

29.24�

2.56k

90.26�

8.27c

7.70�

0.70hi

238.49�

23.27e

Can

dida

quercitrusa

P6

Single

ND

250.25�

29.76fg

1.81�

0.17h

5.60�

0.68def

74.03�

6.61jk

118.95�

10.54b

4.83�

0.47i

230.17�

21.67ef

Co-fermen

ted

0.90�

0.06fg

285.25�

36.58ef

10.83�

0.93bc

8.41�

0.52b

515.07�

47.77c

162.56�

15.45a

48.71�

4.63bc

467.01�

45.59b

Pichia

kluyveriP11

Single

2.20�

0.28c

686.06�

60.21c

3.32�

0.38gh

4.42�

0.45fg

471.64�

42.11c

84.78�

9.30cd

66.78�

6.59a

212.27�

19.72ef

Co-fermen

ted

1.08�

0.12efg

247.64�

23.40efg

8.57�

0.70d

5.79�

0.55def

412.30�

46.01d

121.91�

13.54b

40.49�

3.90d

335.32�

32.28cd

Han

seniasporavineaeS7

Single

1.00�

0.06fg

694.30�

73.81c

3.10�

0.23gh

2.18�

0.20h

149.29�

13.68hi

26.26�

2.71f

3.65�

0.52i

35.74�

3.41i

Co-fermen

ted

1.01�

0.10fg

583.06�

52.24c

3.86�

0.31fg

5.56�

0.64def

205.56�

24.44gh

62.76�

6.71de

5.58�

0.47i

175.81�

15.20efg

H.uvarum

S8

Single

2.63�

0.22b

1043.53�

94.76b

6.85�

0.71e

4.72�

0.46f

199.53�

18.79gh

83.69�

8.55cd

28.63�

2.66e

179.64�

16.95efg

Co-fermen

ted

1.56�

0.14d

491.05�

45.18d

6.35�

0.60e

5.06�

0.55ef

276.87�

26.44f

66.93�

7.27cde

20.13�

1.70f

161.61�

14.84fgh

Can

dida

railenensisS18

Single

1.99�

0.15c

293.33�

29.63ef

3.61�

0.41fgh

6.49�

0.72de

127.00�

11.39ij

164.10�

16.13a

15.71�

1.53fg

376.46�

40.18cd

Co-fermen

ted

0.99�

0.06fg

225.34�

24.35efg

6.61�

0.68e

3.08�

0.28h

236.73�

23.39fg

82.01�

8.03cd

17.99�

1.62fg

202.93�

18.12ef

Metschnikow

iapu

lcherrimaS36

Single

1.40�

0.12de

613.13�

54.13c

9.24�

0.82d

7.89�

0.90bc

190.88�

16.39gh

182.13�

16.62a

12.83�

1.24gh

342.71�

29.54cd

Co-fermen

ted

1.16�

0.10ef

331.25�

38.62ef

19.06�

2.18a

8.27�

0.79b

674.46�

67.03a

162.26�

15.00a

52.33�

4.77b

460.19�

46.58b

Eth

yl

dec

anoate

Eth

yl

nonan

oate

Eth

yl9-

dec

enoate

Methyl

salicy

late

Eth

yl

phen

ylace

tate

2-Phen

yleth

yl

acetate

Eth

yl

dodec

anoate

Eth

yl

hex

adec

anoate

Saccharom

yces

cerevisiae

W3

Single

544.15�

47.64a

6.82�

0.73efg

57.76�

5.33a

8.23�

0.78defg

4.62�

0.37ab

205.31�

19.40d

153.90�

14.55a

4.70�

0.45ef

Wickerham

omyces

anom

alus

JK04

Single

100.30�

7.39h

9.57�

0.92de

12.68�

1.24h

6.79�

0.65efg

4.39�

0.45ab

73.01�

7.12def

37.01�

3.72hi

2.81�

0.27gh

Co-fermen

ted

322.06�

32.90d

8.44�

1.01defg

45.94�

5.13bcd

8.18�

0.69defg

3.92�

0.31b

152.87�

14.28def

115.60�

11.09b

6.23�

0.60de


08

Single

95.26�

9.27h

9.94�

0.89d

10.36�

1.08h

8.56�

0.91cdefg

4.75�

0.50ab

50.87�

5.27ef

26.15�

2.89i

1.74�

0.21h

Co-fermen

ted

271.28�

26.78e

7.89�

0.74defg

26.35�

2.30fg

9.76�

0.93cd

4.11�

0.36b

129.73�

11.93def

78.88�

7.06cde

5.69�

0.53de

Starmerella

bacillarisMR35

Single

7.04�

0.78i

6.38�

0.54efg

ND

14.05�

1.34b

4.07�

0.34b

15.32�

1.73f

8.41�

0.86j

2.42�

0.23gh

Co-fermen

ted

191.63�

18.65fg

6.84�

0.66efg

30.17�

2.99fg

8.25�

0.80defg

4.10�

0.35b

49.83�

4.74ef

59.80�

6.35efg

7.02�

0.68d

Can

dida

quercitrusa

P6

Single

262.87�

27.64e

25.74�

2.54a

27.62�

2.67fg

6.49�

0.63fg

2.97�

0.30c

44.10�

4.17ef

65.51�

6.10def

3.93�

0.33fg

Co-fermen

ted

367.52�

34.19c

6.16�

0.59fg

42.73�

4.11cd

9.18�

0.95cde

3.84�

0.37b

169.12�

14.67de

123.32�

11.74b

8.93�

0.99c

Pichia

kluyveriP11

Single

198.58�

17.48fg

25.34�

2.69a

23.23�

2.03g

15.74�

1.47a

4.40�

0.41ab

1162.86�

117.00b

50.88�

5.97fgh

3.73�

0.36fg

Co-fermen

ted

272.33�

26.04e

5.55�

0.45g

38.42�

3.89de

9.30�

0.88cde

3.99�

0.52b

183.71�

19.66de

94.14�

9.89c

8.81�

0.79c

Han

seniasporavineaeS7

Single

98.81�

7.82h

1.05�

0.09h

32.83�

2.95ef

6.82�

0.65efg

5.29�

0.49a

1906.57�

174.09a

11.31�

1.04j

2.96�

0.28gh

Co-fermen

ted

246.04�

22.87ef

5.92�

0.57fg

51.22�

4.65ab

c9.23�

0.93cde

3.83�

0.36b

709.09�

69.51c

70.95�

6.00de

14.06�

1.36a

H.uvarum

S8

Single

88.95�

11.02h

9.12�

0.88def

16.49�

1.49h

8.71�

0.80cdef

4.83�

0.45ab

189.39�

16.41de

47.75�

4.42gh

6.34�

0.60d

Co-fermen

ted

160.78�

15.12g

5.31�

0.48g

34.32�

3.50ef

6.17�

0.67g

3.73�

0.34b

108.90�

10.58def

75.28�

7.76de

13.15�

1.19a

Can

dida

railenensisS18

Single

227.53�

20.89ef

17.58�

1.72c

43.60�

4.72cd

9.17�

0.89cde

4.40�

0.40ab

52.01�

4.77ef

83.75�

7.36cd

5.83�

0.60de

Co-fermen

ted

206.97�

20.45fg

6.34�

0.56efg

50.23�

5.53ab

c6.71�

0.71efg

4.40�

0.39ab

90.56�

8.70def

69.03�

6.79de

7.28�

0.71d

Metschnikow

iapu

lcherrimaS36

Single

418.12�

39.25b

21.06�

2.17b

53.18�

4.30ab

8.60�

0.84cdefg

1.42�

0.17c

79.56�

8.00def

122.56�

11.20b

3.97�

0.43fg

Co-fermen

ted

377.49�

34.97bc

6.71�

0.62efg

46.38�

4.74bcd10.83�

1.04c

2.89�

0.30c

179.69�

16.07de

120.50�

11.03b

10.99�

1.14b



ificantdifference




DiscussionMost non-Saccharomyces yeasts are known for their low etha-nol tolerance because of chemical stress induced by ethanolaccumulation, such as damage to the plasma membrane,which results in low ethanol productivity or prolonged fer-mentation time (Thomas et al. 1978, Beavan et al. 1982,Ingram and Buttke 1984, Pina et al. 2004). Some researchers,however, have reported that Hanseniaspora, Candida andKluyveromyces species have a similar ethanol tolerance to thatof S. cerevisiae (Mills et al. 2002, Pina et al. 2004, Xufre et al.2006, Nisiotou et al. 2007, Fleet 2008). In contrast, otherstudies have revealed that some non-Saccharomyces yeastshave a high ethanol tolerance; however, their growth wasinhibited by the antagonistic effect of S. cerevisiae when usedfor fermentation (Jolly et al. 2003, Rodríguez et al. 2010,Sadoudi et al. 2012). In the present study, H. vineae andS. bacillaris showed interesting responses among all species.Hanseniaspora vineae showed a much faster fermentation ratecompared to that of other species when used for wine fer-mented with individual species. In addition, H. vineaemaintained a high viable cell count until the end of fermenta-tion when used for co-fermentation with S. cerevisiae, whichsuggests that H. vineae affected the production of winesecondary metabolites. Starmerella bacillaris initiated alcohol

fermentation earliest but was the last to complete amongsingle-fermented wines. This is likely because this species hasa fructophilic character, which implies that it first consumesfructose, and then slowly utilises glucose, thus explaining theprolonged fermentation period of 24 days (Sadoudiet al. 2012).

pH and TA are important factors for wine quality, suchas taste, aroma and sugar/acid balance of wines, which areaffected by the balance between anionic forms of organicacids (primarily tartaric acid and malic acid) and the majorcations (primarily K) (Boulton 1980, Ohtsuki et al. 2001,Kodur 2011). In our study, each non-Saccharomyces yeastshowed different metabolic behaviours during alcohol fer-mentation. The pH and TA of most single-fermented winesgradually changed, but were found to be higher and lower,respectively, than those of co-fermented wines. Tartaric acidconcentration did not differ significantly among the winesbecause this acid is less responsive to breakdown by micro-organisms during alcohol fermentation (Butzke 2010). Malicacid concentration of wines fermented by single-culture ofW. anomalus and H. uvarum was lower than that of the con-trol and co-fermented wines, while that of wine fermentedby H. vineae was higher than that of the control and co-fermented wines. Although acetic acid concentration ofsingle-fermented wines was higher than that of the controland co-fermented wines, the values remained lower thanthe odour threshold (0.7–1.0 g/L) (Lambrechts and Pretorius2000). We assumed that different pH values and concentra-tion of organic acids by each non-Saccharomyces yeast cancreate distinct sensory properties of the final wine.

Phenolic substances are one of the most important fac-tors for the sensory properties of wine, such as colour,astringency, bitterness and effective antioxidant properties(Paixão et al. 2007). In our study, the concentration of phe-nolic substances did not show significantly different patternsbetween single- or co-fermented wines; however, the con-centration of phenolic substances in co-fermented wineswas slightly higher than that of single-fermented wines.

Volatile aromatic compounds, such as esters, terpenes,acids, alcohols and phenols, of wine are one of the mostimportant factors that establish differences in the variousstyles of wines made throughout the world. Thus, modulat-ing wine flavour by various microorganisms, such as non-Saccharomyces yeasts or malolactic bacteria, has beenhighlighted for the last few decades (Swiegers et al. 2005).Among various aromatic compounds, higher alcohols, alsoknown as fusel alcohols, quantitatively account as thelargest group of aromatic compounds in wines. Excessive

Figure 6. Profiling of volatile esters based on principal component analysis(PCA) and solid-phase microextraction (SPME)/GC/MS analysis of CampbellEarly grape wine fermented by individual species of non-Saccharomycesyeasts ( ) and co-fermented by individual species of non-Saccharomycesyeasts together with S. cerevisiae W-3 ( ). S. cerevisiae W-3 ( );Wickerhamomyces anomalus JK04 ( , ); Torulaspora delbrueckii JK08( , ); Starmerella bacillaris MR35 ( , ); Candida quercitrusa P6 ( , );Pichia kluyveri P11 ( , ); Hanseniaspora vineae S7 ( , ); H. uvarum S8( , ); Candida railenensis S18 ( , ); and Metschnikowia pulcherrimaS36 ( , ).

Table 4. Hue and intensity values of Campbell Early wines fermented with individual non-Saccharomyces yeasts and co-fermented with individual non-Sac-charomyces yeasts together with S. cerevisiae.

Hue Intensity

Inoculated yeast Single-fermentation Co-fermentation Single-fermentation Co-fermentation

Saccharomyces cerevisiae W3 0.985 � 0.012i ND 5.011 � 0.015a NDWickerhamomyces anomalus JK04 1.254 � 0.008d 1.062 � 0.022gh 3.712 � 0.027g 4.684 � 0.012bcTorulaspora. delbrueckii JK08 1.504 � 0.005a 1.179 � 0.028e 3.296 � 0.041h 4.288 � 0.012eStarmerella bacillaris MR35 1.360 � 0.007c 1.136 � 0.006ef 3.223 � 0.090h 4.485 � 0.015dCandida quercitrusa P6 1.119 � 0.018efg 1.006 � 0.017hi 4.554 � 0.060cd 4.310 � 0.043ePichia kluyveri P11 1.109 � 0.016fg 0.998 � 0.032hi 4.199 � 0.032e 4.751 � 0.039bHanseniaspora vineae S7 1.449 � 0.010b 1.105 � 0.026fg 3.675 � 0.027g 4.162 � 0.006eH. uvarum S8 1.142 � 0.018ef 1.018 � 0.029hi 4.302 � 0.050e 4.691 � 0.035bcCandida railenensis S18 1.113 � 0.004fg 1.031 � 0.030hi 4.177 � 0.101e 4.551 � 0.052cdMetschnikowia pulcherrima S36 1.445 � 0.024b 1.056 � 0.024gh 3.831 � 0.031f 4.566 � 0.052cd

Different letters within the same items indicate a significant difference (P < 0.05). ND, not detected.



concentration of higher alcohols can lead to a negativeimpact on wine aroma, such as a strong, pungent smell andtaste, whereas an appropriate concentration (below300 mg/L) of these compounds can contribute to positiveeffects on wine aroma, such as fruity characters(Lambrechts and Pretorius 2000, Swiegers et al. 2005).Esters are also the most abundant aromatic compounds inwine and are known as one of the secondary metabolitegroups produced by various non-Saccharomyces yeasts duringfermentation (Peddie 1990, Lambrechts and Pretorius2000). Different esters frequently have a synergistic effecton flavour profiles and influence each flavour well belowtheir individual threshold concentration. Because the con-centration of most esters during fermentation is near theirthreshold value, small changes can result in a significanteffect on wine aroma (Sumby et al. 2010). Because higheralcohols are important precursors for ester synthesis duringfermentation (Soles et al. 1982), controlling the appropriatebalance between higher alcohols and esters in the final wineand understanding the fermentation traits of individualnon-Saccharomyces yeasts are required. In our study, all thesingle-fermented wines had a different composition of vola-tile higher alcohol and ester compounds. In particular, aconsiderably high quantity of ethyl acetate and2-phenylethyl acetate was detected in wines fermented byW. anomalus and H. vineae, respectively. Thus, it is necessaryto select a suitable ratio between these species andS. cerevisiae to avoid the negative effect of excessively highconcentration of ethyl acetate (Lilly et al. 2000, Rojas et al.2001, Erten 2002). The PCA shows distinguishable patternsof both volatile higher alcohols and esters between single-and co-fermented wines. The volatile esters in co-fermentedwines, however, were more widely distributed comparedwith the volatile higher alcohols in co-fermented wines,suggesting that co-fermentation with non-Saccharomycesyeasts and S. cerevisiae can contribute to the formation of acomplicated and more complex pattern of aromatic estercompounds instead of higher alcohols, ultimately affectingwine quality.

Hue and intensity values of wine are important parame-ters that determine the sensory quality of wine, and theyare affected by change in the pH and alcohol concentrationthat occur during alcohol fermentation (Coradini et al.2014). In our study, some single-fermented wines showedrelatively high hue and low intensity values compared withthat of the Control and co-fermented wines; these resultsfor the visual attributes of the final wines could be becauseof the pH increase caused by some of the inoculated species.

The sensory evaluation scores of all wines were not sig-nificantly different. Wines fermented by non-Saccharomycesyeasts gained slightly higher flavour and overall preferencescores. In contrast with the results for the volatile aromaticcompounds and for sensory evaluation, the flavour scores ofwines fermented by a single-culture of W. anomalus and asingle- and co-culture of H. uvarum, which had a high con-centration of ethyl acetate, were notably higher than thoseof the Control wine. Additionally, both wines fermented bysingle- and co-culture of M. pulcherrima received good scoresby increasing the ethyl acetate concentration during fer-mentation. The flavour score of wine fermented by a single-culture of H. vineae was significantly lower than that ofother wines because of the excessive concentration of2-phenylethyl acetate. Wines fermented by P. kluyveri and aco-culture of H. vineae, however, gained good scores becauseof the appropriate increase in 2-phenylethyl acetate. Inaddition, other wines that did not show greatly changedconcentration of ethyl acetate and of 2-phenylethyl acetatereceived also positive flavour scores because of interactionsbetween other volatile aromatic compounds. This indicatesthat single volatile aromatic compounds such as ethyl ace-tate and 2-phenylethyl acetate can improve wine quality;however, the synergistic effect of overall aromatic com-pounds is more important to wine aroma.

ConclusionsThe present work revealed variable fermentation kineticsfor each species during fermentation and a variable relation-ship of volatile higher alcohol and ester compounds

Table 5. Sensory scores for Campbell Early wines fermented with individual non-Saccharomyces yeasts and co-fermented with individual non-Saccharomy-ces yeasts together with S. cerevisiae.

Sensory evaluation score

Inoculated yeast Inoculationstrategy

Colour Flavour Sweetness Sourness Overallpreference

Saccharomyces cerevisiae W3 Single 6.95 � 1.82ab 6.15 � 1.66ab 4.20 � 1.70a 5.25 � 1.74a 5.35 � 1.69aWickerhamomyces anomalus

JK04Single 5.94 � 1.76ab 6.39 � 1.89ab 4.68 � 1.48a 5.40 � 2.02a 5.67 � 1.78aCo-fermented 7.00 � 1.89ab 5.70 � 1.49ab 4.25 � 2.31a 5.55 � 1.96a 5.30 � 1.87a

Torulaspora delbrueckii JK08 Single 6.66 � 1.44ab 5.31 � 1.37ab 4.05 � 1.74a 5.85 � 1.74a 5.40 � 1.94aCo-fermented 7.05 � 1.43ab 5.80 � 2.29ab 3.95 � 1.76a 5.50 � 1.54a 4.95 � 1.50a

Starmerella bacillaris MR35 Single 5.67 � 1.68b 5.76 � 1.71ab 4.68 � 1.48a 5.67 � 2.05a 5.49 � 1.70aCo-fermented 6.70 � 1.66ab 5.80 � 1.84ab 4.20 � 1.82a 5.25 � 1.74a 5.20 � 1.54a

Candida quercitrusa P6 Single 7.47 � 1.21a 5.85 � 1.64ab 3.69 � 1.37a 5.31 � 1.89a 4.59 � 1.49aCo-fermented 7.20 � 1.54ab 6.50 � 1.79ab 4.40 � 1.90a 4.55 � 1.99a 5.80 � 1.64a

Pichia kluyveri P11 Single 6.75 � 1.15ab 5.94 � 1.86ab 4.14 � 1.76a 6.39 � 1.49a 5.40 � 1.65aCo-fermented 6.95 � 1.54ab 5.90 � 1.77ab 3.95 � 1.88a 4.95 � 1.57a 5.00 � 1.75a

Hanseniaspora vineae S7 Single 5.85 � 1.42ab 4.68 � 1.23b 4.68 � 1.59a 5.31 � 1.60a 4.50 � 1.60aCo-fermented 7.30 � 1.38ab 6.00 � 1.72ab 3.75 � 1.94a 5.00 � 2.08a 5.30 � 1.78a

H. uvarum S8 Single 7.29 � 1.24ab 6.57 � 1.21a 4.95 � 1.42a 5.13 � 1.34a 5.58 � 1.15aCo-fermented 7.20 � 1.28ab 6.45 � 1.70ab 4.00 � 2.13a 5.30 � 2.30a 5.25 � 2.15a

Candida railenensis S18 Single 7.47 � 1.46a 5.76 � 1.25ab 3.69 � 1.24a 5.58 � 1.53a 4.50 � 1.37aCo-fermented 7.30 � 1.49ab 5.90 � 1.83ab 3.90 � 1.80a 5.25 � 2.00a 5.10 � 1.83a

Metschnikowia pulcherrimaS36

Single 6.30 � 1.37ab 6.21 � 1.37ab 4.05 � 1.29a 5.22 � 1.64a 5.22 � 1.53aCo-fermented 7.25 � 1.48ab 6.15 � 1.93ab 3.60 � 1.93a 5.60 � 2.19a 4.95 � 2.11a

Different letters within the same column indicate a significant difference (P < 0.05).



between single- and co-fermented wines using nine speciesof non-Saccharomyces yeasts. Different fermentation kineticsmight aid in understanding the use of each species for winefermentation. Furthermore, analysis of volatile aromaticcompounds suggested the importance of synergistic effectsamong those compounds to enhance wine quality, whichshows the possibility of developing various types of distinc-tive wines using different non-Saccharomyces yeasts.

AcknowledgementThis work was supported by the National Research Founda-tion of Korea, Republic of Korea under Research Grant(NRF-2017R1D1A3B03033451).

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Manuscript received: 21 January 2019

Revised manuscript received: 18 April 2019

Accepted: 5 May 2019



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