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

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

Early wines

S.-B. LEE¹, C. BANDA¹and H.-D. PARK^1,2

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

Corresponding author: Dr Heui-Dong Park, email hpark@knu.ac.kr

Abstract

Background and Aims: Wine made from the Campbell Early grape cultivar has less ﬂavour than wine made from European grape cultivars. The aim of this study was to investigate the individual fermentation characteristics of several non- Saccharomycesyeasts 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-fermentation with Saccharomyces cerevisiae. Several fermentation characteristics and physiological properties were investigated. Volatile higher alcohol and ester compounds were also analysed by GC/MS and by principal component analysis. Single-fermented wines showed different fermentation kinetics, whereas co-fermented wines showed similar, but slightly different, fermentation kinetics depending on their ethanol tolerance. Principal component analysis indicated that the composition of both volatile higher alcohols and esters was distinguishable between single and co-fermented wines, but volatile esters in co-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-fermentation than volatile higher alcohols, which were produced during the early phase of fermentation. Moreover, interactions among the various volatile aromatic compounds affected theﬁnal wine aroma.

Signiﬁcance of the Study: These ﬁndings could provide valuable information to researchers and winemakers on the enhancement of wine aroma using non-Saccharomycesyeasts.

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

Introduction

Wine has a long history and is made by a complex interaction among yeasts, lactic acid bacteria and fungi (Heard and Fleet 1985, Zagorc et al. 2001). Most Korean winemakers have used Saccharomyces cerevisiaeas a wine starter owing to its high ethanol productivity and ability to protect wine from unfavourable microorganisms during fermentation (Philliskirk and Young 1975, Casey and Ingledew 1986).

Campbell Early grape, the predominant cultivar grown in Korea, is not competitive with the European grape cultivars for wine fermentation because of its high malic acid concentration caused by early harvesting to enhance grape colour (Lee et al. 2016a). Moreover, chaptalisation is necessary for winemaking since the cultivar has low TSS (14– 15Brix), which results in a wine of low ﬂavour (Hwang and Park 2009). To improve wine quality or to reduce malic acid concentration, several strategies, such as MLF, mixing with different grape cultivars, carbonic maceration and co- fermentation with non-Saccharomyces yeasts, have been attempted (Pozo-Bayón et al. 2005, Yook et al. 2007, Chang et al. 2011, Sadineni et al. 2012, Hong and Park 2013, Hu et al. 2016, Lee et al. 2016b). Among these approaches, inoculation of grape must with non-Saccharomycesyeasts has been widely utilised.

Non-Saccharomycesyeasts have been considered of minor signiﬁcance or as spoilage yeasts for a long time, since some

species such asHanseniasporaand Zygosaccharomycesproduce an excessive concentration of acetic acid during fermentation (Loureiro and Malfeito-Ferreira 2003, Padilla et al.

2016). Several recent studies, however, have reported that non-Saccharomyces yeasts can contribute to an improvement of wine quality by producing aromatic compounds, such as esters, terpenes, acids and higher alcohols, during the early stage 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 as grape cultivar, climate conditions, localisation, speciﬁc weather conditions in each year (vintage), and ripening phase, which can intimately inﬂuence quality and distinc- tion of local wines (Querol et al. 1990, Schütz and Gafner 1993, Jolly et al. 2006, Brilli et al. 2015). In addition, some researchers have indicated that indigenous yeasts can contribute 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 and Gafner 1993, Mercado et al. 2007, Hong and Park 2013).

Among the volatile aromatic compounds of wine fermented by yeasts, higher alcohols are the largest group and play an important role as ester precursors (Soles et al. 1982, Lambrechts and Pretorius 2000). They are typically classiﬁed into: (i) the aliphatic alcohol group (e.g. propanol, isoamyl alcohol, isobutanol and active amyl alcohol) and; (ii) the doi: 10.1111/ajgw.12405

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aromatic alcohol group (e.g. 2-phenylethyl alcohol and tyrosol) (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 of higher alcohols is below 300 mg/L and exceeds 400 mg/L, respectively. Esters, a group of the most plentiful compounds in wine, comprise two major groups affecting the flavour of fermented beverages: (i) acetate esters, such as ethyl acetate, isoamyl acetate, iso-butyl acetate, 2-phenylethyl acetate and hexyl acetate, which are composed of acetate and ethanol (or a complicated alcohol derived from amino acid metabolism), and are synthesised by acetyltransferase (AAT) using alcohol and acetyl-CoA and (ii) ethyl esters, such as ethyl hexanoate, ethyl octanoate and ethyl decanoate, which are composed of ethanol and a medium-chain fatty acid (MCFA), and are formed by two mechanisms, including esterification catalysed by fatty acid ethyl ester synthases/carboxylesterases and alcoholysis catalysed by acyl-CoA: ethanol O-acyltransferases (Rojas et al. 2001, Saerens et al. 2006, 2008, Padilla et al. 2016, Hu et al. 2018). For the last few decades, the impact of these compounds on the composition of wine aroma and their mechanisms of formation in non-Saccharomyces yeasts have been revealed by many researchers. Nevertheless, most researchers have focused on co-fermentation with non-Sac- charomyces yeasts and S. cerevisiae, and few have studied the effect of individual non-Saccharomycesyeasts on fermentation of grape juice. Therefore, understanding the fermentation characteristics of each non-Saccharomycesyeast and their effect onfinal wine aroma are still necessary for improving wine quality.

In this study, we investigated the changes in several fermentation characteristics and physicochemical properties of Campbell Early wines fermented by nine species of previously isolated indigenous non-Saccharomyces yeasts. Inoculation strategies, such as fermentation with single non-Saccharomyces yeasts and co-fermentation with non-Saccharomycesyeasts and S. cerevisiae, were investigated for wine fermentation to reveal the distinctive characteristics derived from each species. Vola- tile aromatic compounds of all wines were analysed using principal component analysis (PCA), in addition, to sensory evaluation of the wines.

Materials and methods Yeast species and growth medium

Saccharomyces cerevisiae W-3, an industrial wine yeast (Yokotsuka and Matsudo 1992), and nine species of non- Saccharomyces yeasts, namely Wickerhamomyces anomalus JK04, Torulaspora delbrueckii JK08, Starmerella bacillaris MR35, Candida quercitrusa P6, Pichia kluyveri P11, Han- seniaspora vineae S7, Hanseniaspora uvarum S8, Candida railenensisS18 andMetschnikowia pulcherrimaS36, were used for wine fermentation. All non-Saccharomycesyeasts used in the present study were previously isolated from food materials, such as nuruk, Muscat Bailey A grape, persimmon and Sémillon grape (Wahyono et al. 2016, Jeong et al. 2017, Mr S.B. Lee, pers. comm., 2019). All strains were cultured in yeast peptone dextrose (YPD) broth (yeast extract 10 g/L, peptone 20 g/L and glucose 20 g/L) at 30C for 24 h prior to the 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-

Saccharomycesyeasts, becauseS. cerevisiaedoes not survive on this medium.

Wine fermentation

Campbell Early grapes (Vitis labruscacultivar) (14.0Brix at harvest), cultivated in Sangju, Korea, were washed, stem- med, crushed and chaptalised to 24Brix to prepare grape must for wine fermentation. To inhibit growth of unfavourable bacteria, 200 mg/L potassium metabisulfite (K2S2O5) was added to the 5 kg grape must before inoculation with wine yeast. For single fermentation, the yeast cells cultured overnight in YPD medium at 30C with shaking (150 rpm) were inoculated into 250 g grape must in 1 L Erlenmeyer flasks. The flasks were incubated at 30C with shaking (150 rpm) for 2 days, until the yeast cells reached

~10⁸CFU/mL. They were then inoculated into 5 kg grape must in a 20 L fermentation container with a vented lid, and each must was fermented at 20C without shaking until fermentation was completed. For fermentation with S. cerevisiae and non-Saccharomycesyeasts, the initial culture was prepared at a ratio of 1:9. For this,S. cerevisiaeand non- Saccharomyces yeast, cultured overnight in YPD medium at 30C with shaking (150 rpm), were inoculated into 25 and 225 g grape must in 100 mL and 1 L Erlenmeyer ﬂasks, respectively, The following procedure was followed as described above. Final wines wereﬁlter-sterilised and stored at 4C for further analysis and sensory evaluation.

Analytical methods

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

Total soluble solids was measured with a refractometer, in accordance with AOAC guidelines (Caputi 1995) and reducing sugar concentration was analysed using dinitrosalicylic acid according to AOAC guidelines (Caputi 1995). Alcohol concentration was measured with a hydrometer based on the speciﬁc gravity of wine distillates [expressed as % (v/v)] at 15C (Caputi 1995). pH was measured with a pH meter (Mettler-Toledo, Schwerzenbach, Switzerland) and TA [expressed as tartaric acid (%)] was determined by titration ofﬁltrates with 0.1 N NaOH.

The concentration of phenolic substances was determined with the Folin–Ciocalteau method (Singleton and Rossi 1965). Hue and intensity values were obtained from OD420/ OD₅₂₀and OD₄₂₀+ OD₅₂₀ (Kim et al. 2008). The concentration of free sugars and of organic acids was determined by HPLC (Model Prominence, Shimadzu, Kyoto, Japan) with a Sugar-Pak I column (diameter 6.5×300 mm; Waters, Mil- ford, MA, USA) and PL Hi-Plex H column (diameter 7.7×300 mm; Agilent Technologies, Santa Clara, CA, USA).

The chromatography conditions for the free sugars were as follows: ﬂow rate, 0.5 mL/min; temperature, 90C and mobile phase, 50 mg/L Ca-ethylenediaminetetraacetic acid (Ca-EDTA) buffer (Kim et al. 2018). The chromatography conditions for the organic acids were as follows: ﬂow rate, 0.6 mL/min; temperature, 65C and mobile phase, 0.005 mol

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

Analysis of volatile aromatic compounds

The volatile aromatic compounds were determined with a 7890A GC/MS (Agilent Technologies) equipped with a flame ionisation detector (FID) (Lee et al. 2016b). The compounds were separated on a DB-WAX column (60 m×250μm×0.25 mm; Waters). The detector was an Agilent Technologies 5975C Inert XL MSD with a Triple- Axis detector. Helium was the carrier gas at a constantflow rate of 1 mL/min. The chromatographic oven was initially held at 40C for 2 min, increased at 2C/min to 220C, increased continuously at 20C/min to 240C, and then maintained at 240C for 5 min. Volatile aromatic compounds were collected with a solid-phase microextraction (SPME) fibre (50/30μm DVB/CAR/PDMS; Supelco, Bell- efonte, PA, USA). The volatile aromatic compounds were extracted from the wine in headspace (HS) mode with magnetic stirring. The sample (5 mL) was placed in an HS vial (20 mm, PTFE/silicon septum, magnetic cap) and 1.25 g of NaCl was added to increase the concentration of volatile aromatic compounds in the HS. Prior to extraction, the sample was shaken in a water bath at 35C for 20 min to achieve equilibrium. Subsequently, the SPME fibre was spiked into the vial and held at 35C for 40 min. Volatile aromatic compounds were identified by a comparison of their GC retention time and MS with spectral data from the Wiley9Nist 0.8 library mass spectral search program, version 5.0 (Torrens et al. 2004).

Sensory evaluation

Aﬁve-point hedonic scale was used for sensory evaluation.

Before being subjected to sensory evaluation, each wine was place in a sample bottle and left undisturbed at room temperature for 1 h with the bottle lid closed. Afterﬂavour evaluation, 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 taste discrimination from the Department of Food Science and Technology, Kyungpook National University, Korea. Each judge evaluated the wines with an at least a 3 min interval between 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 analysis

All experiments were in triplicate, and results were analysed with SAS software (version 9.4, SAS Institute, Cary, NC, USA). Signiﬁcance was determined at a threshold of P< 0.05 using ANOVA, followed by the Duncan’s multiple range test (Kim et al. 2008). Similarities were determined among 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 software. In addition, correlation coefﬁcients of volatile compounds identiﬁed by SPME/GC/MS were analysed by Pearson’s correlation analysis (Chung et al. 2016).

Results

Fermentation characteristics of Campbell Early wine Figure 1 shows the TSS, and concentration of reducing sugars and of alcohol of Campbell Early wine fermented individually withS. cerevisiaeand each of the nine species of non-Saccharomyces yeasts isolated previously and co- fermented with each of those strains together with S. cerevisiae.The alcohol fermentation of wine co-fermented and fermented with S. cerevisiae (control) was completed within 7 days, and the alcohol concentration of these wines reached 12.4–13.0%. Wine fermented, however, with each of the nine species of non-Saccharomyces yeasts showed fermentation kinetics that depended on the species. Alcohol fermentation by S. bacillaris occurred as the earliest but slowest among all single-fermented wines with non-Saccha- romyces yeasts (23 days for fermentation). Hanseniaspora vineae began to produce alcohol on day 2 of fermentation and completed fermentation on day 11, which was the fastest among all single-fermented wines. Other non-

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Figure 1. Effect of fermentation of Campbell Early grape must with either Saccharomyces cerevisiae W-3 and or a single strain of several non- Saccharomycesspecies ( , , ) and of co-fermentation withS. cerevisiae W-3 together with one of several non-Saccharomycesspecies ( , , ) on the 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) Hanseniaspora vineae S7; (h) H. uvarum S8; (i) Candida railenensis S18; and (j)Metschnikowia pulcherrimaS36.

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Saccharomyces yeasts began alcohol fermentation at days 7–11 and completed fermentation at days 15–23. The pH and TA of wines fermented with S. cerevisiae, such as the Control and co-fermented wines, rapidly decreased and increased in the early phase of the fermentation, andﬁnally reached 3.50–3.58 and 0.51–0.58%, respectively (Figure 2).

In contrast, the kinetics of wines fermented with a single species varied depending on the species. The TA of wines fermented withW. anomalus and T. delbrueckii signiﬁcantly increased during the early phase of fermentation and reached a maximum value at the middle phase; the pH of these wines decreased throughout the early phase of fermentation and slightly increased after the middle phase. The TA and pH of wine fermented with S. bacillaris sharply increased and decreased, respectively, at the beginning of fermentation and gradually decreased and increased throughout the middle and latter phases. The TA and pH of wine fermented withH. vineaedid not change notably compared to that of other wines. Moreover, the TA and pH of

wines fermented by the other individual species steadily increased and decreased, respectively, throughout fermentation. Generally, the pH of wines fermented with a single species was higher than that of wines co-fermented, whereas TA of wines fermented with a single species, except forW. anomalus, was lower than that of wines co-fermented.

Viable cell counts of wine fermented with H. uvarum and C. railenensis decreased considerably in the early phase of fermentation and increased signiﬁcantly after day 5 of fermentation (Figure 3), whereas those of other wines fermented with a single species slightly decreased at the beginning, increased again, and were maintained at

>7.5 log CFU/mL. For co-fermented wines, the viable cell count of non-Saccharomycesyeasts, except forS. bacillarisand H. vineae, rapidly decreased after the middle phase of fermentation since alcohol concentration was produced and those species, except forW. anomalus, completely died when fermentation was completed. Moreover, that of S. bacillaris MR35 somewhat decreased when the wine alcohol concentration reached 12%. Conversely, that ofH. vineaeremained at a high level throughout fermentation, implying that those two species have an ethanol tolerance higher than that of other non-Saccharomycesyeasts. The concentration of phenolic substances of wines fermented with a single species of non-Saccharomyces yeasts, except for S. bacillaris, increased until the middle phase of fermentation and then decreased gradually, whereas that of S. bacillarisdid not change notably during alcohol fermentation (Figure 4). The concentration of phenolic substances of all co-fermented wines generally increased throughout alcohol fermentation.

Concentration of free sugars and organic acids in Campbell Early wine

Control and co-fermented wines consumed glucose and sucrose completely (Table 1). In addition, a low concentration of fructose was detected. In most wines fermented with a single species, some free sugars, such as glucose, sucrose and fructose, were not completely consumed; the concentration of fructose of wines fermented with C. quercitrusa, P. kluyveriandH. vineaewas higher than that in other wines.

The concentration of citric acid of wines fermented with each of the non-Saccharomyces yeasts, except for W. anomalus, was lower than that of co-fermented wines.

Furthermore, the concentration of malic acid of wines fermented with W. anomalus and H. uvarum was the lowest, and that of wine fermented by H. vineae was the highest among all wines. The concentration of succinic acid of all wines fermented with non-Saccharomyces yeasts, except for S. bacillaris, was lower than that of co-fermented wines, whereas the concentration of tartaric acid and lactic acid was not signiﬁcantly different between the wines. Acetic acid concentration of wines fermented with individual species was slightly higher than that of Control and co- fermented wines.

Volatile aromatic compounds of Campbell Early wine Eight volatile higher alcohols were detected in all wines (Table 2), with each alcohol showing a variable pattern depending on the species and inoculation strategy. The concentration of 1-propanol of wines single-fermented with T. delbrueckii,P. kluyveri,H. uvarumandC. railenensiswas sig- niﬁcantly higher than that of co-fermented wines. Further- more, the concentration of 1-propanol of some wines fermented by other species was slightly higher than that of

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Figure 2. Effect of fermentation of Campbell Early grape must with either Saccharomyces cerevisiae W-3 and or a single species of several non- Saccharomycesyeasts ( , ) and of co-fermentation withS. cerevisiaeW-3 together with one of several non-Saccharomycesspecies ( , ) on the pH ( , ) and TA ( , ). (a) S. cerevisiae W-3; (b) Wickerhamomyces anomalusJK04; (c)Torulaspora delbrueckiiJK08; (d)Starmerella bacillaris MR35; (e) Candida quercitrusa P6; (f) Pichia kluyveri P11;

(g)Hanseniaspora vineaeS7; (h)H. uvarumS8; (i)Candida railenensisS18;

and (j)Metschnikowia pulcherrimaS36.

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co-fermented wines. Iso-butanol concentration of wines fermented by single-culture of S. bacillaris, C. railenensis, M. pulcherrima and T. delbrueckii was signiﬁcantly higher than that of co-fermented wines. Iso-amyl alcohol concentration of wines fermented by both single- and co-culture of C. railenensis was the highest among each inoculation type, while those of wines fermented by both single- and co-culture ofS. bacillariswere the lowest among each inoculation type. The concentration of 1-hexanol in wine fermented by non-Saccharomyces yeasts, except for P. kluyveri, was higher than that of co-fermented wines. Fur- thermore, the concentration of 1-heptanol of co-fermented wines by non-Saccharomyces yeasts, except for C. railenensis andM. pulcherrima, was higher than that of wines fermented with individual yeasts. In addition, 1-heptanol concentration of wines fermented by both single- and co-culture of H. uvarum and C. railenensis was higher than that of the

control wine. Thus, most non-Saccharomyces yeasts do not produce 1-heptanol during fermentation. The concentration of 2-ethyl-1-hexanol of wines fermented by individual non- Saccharomyces yeasts was higher than that of co-fermented and Control wines, whereas 1-octanol concentration of wines fermented by individual non-Saccharomyces yeasts, except forH. uvarum, was lower than that of co-fermented wines. The concentration of 1-nonanol of all wines, except for single-culture of H. vineae, was lower than that of the Control wine. Additionally, 1-nonanol concentration of single-fermented wines by non-Saccharomyces yeasts, except forH. vineae, was lower than that of co-fermented wines.

Principal component analysis of volatile higher alcohols of all wines indicated that PC1 and PC2 accounted for 51.21 and 27.30% of the variance, respectively (Figure 5). The PCA 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 either Saccharomyces cerevisiae W-3 and or a single species of several non- Saccharomycesyeasts ( ) and of co-fermentation withS. cerevisiaeW-3 ( ) together with one of several non-Saccharomycesspecies ( ) on the viable cell count ( , , ). (a) S. cerevisiae W-3; (b) Wickerhamomyces anomalusJK04; (c)Torulaspora delbrueckiiJK08; (d)Starmerella bacillaris MR35; (e) Candida quercitrusa P6; (f) Pichia kluyveri P11;

(g)Hanseniaspora vineaeS7; (h)H. uvarumS8; (i)Candida railenensisS18;

and (j)Metschnikowia pulcherrimaS36.

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Figure 4. Effect of fermentation of Campbell Early grape must with either Saccharomyces cerevisiae W-3 and or a single species of several non- Saccharomycesyeasts ( ) and of co-fermentation withS. cerevisiaeW-3 together with one of several non-Saccharomyces species ( ) on the concentration 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) Pichia kluyveriP11; (g)Hanseniaspora vineaeS7; (h)H. uvarumS8; (i)Candida railenensisS18; and (j)Metschnikowia pulcherrimaS36.

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Table1.ConcentrationoffreesugarsandorganicacidsinCampbellEarlywinesfermentedwithindividualnon-Saccharomycesyeastsandco-fermentedwithindividualnon-SaccharomycesyeaststogetherwithS.cerevisiae. Freesugarsconcentration(g/L)Organicacidconcentration(g/L) InoculatedyeastInoculation strategyGlucoseSucroseFructoseCitricacidTartaric acidMalicacidSuccinicacidLacticacidAceticacid Saccharomyces cerevisiaeW3SingleNDND0.360.01cd1.050.03efgh1.840.14a3.120.11bcd1.180.08defg0.190.00d0.030.01gh Wickerhamomyces anomalusJK04Single0.080.02a0.040.00a0.560.02cd1.330.06c1.810.13a2.660.09e1.040.09fgh0.220.01bcd0.450.03a Co-fermentedNDND0.400.02cd1.150.04de2.100.09a3.330.09b1.330.10bcde0.230.0bc0.030.01gh Torulaspora delbrueckiiJK08Single0.060.01ab0.040.01a0.660.02cd0.820.03j1.750.16a3.070.10bcd0.950.08gh0.240.02b0.200.02d Co-fermentedNDND0.720.02c0.980.02fghi1.930.12a2.540.12e1.420.11bcd0.230.00bc0.040.01fgh Starmerella bacillarisMR35SingleNDND0.320.01d0.900.05ij1.810.15a3.160.12bcd1.330.12bcde0.210.01bcd0.290.04b Co-fermentedNDND0.360.01cd1.060.05efg1.950.14a2.980.07cd1.090.09efgh0.200.01cd0.060.01fg Candidaquercitrusa P6Single0.080.01a0.010.01b1.530.13b0.940.04ghij1.680.18a3.310.14b0.990.09gh0.220.01bcd0.140.02e Co-fermentedNDND0.450.01cd1.160.03de1.990.10a3.240.09bc1.280.08cdef0.220.01bcd0.020.01gh PichiakluyveriP11Single0.070.01a0.030.00ab1.490.08b0.880.06ij1.780.15a3.330.09b0.830.08h0.190.00d0.260.03c Co-fermentedNDND0.380.02cd1.210.04d2.030.13a3.320.13b1.460.13bc0.230.02bc0.020.01gh Hanseniaspora vineaeS7SingleND0.030.01ab7.250.56a1.100.10edf1.840.19a4.270.17a1.050.09fgh0.200.02cd0.160.02e Co-fermentedNDND0.400.01cd1.610.08a1.930.09a2.970.06cd1.270.12cdef0.200.01cd0.040.01fgh H.uvarumS8Single0.070.01a0.020.01ab0.360.01cd0.840.06ij1.720.14a2.690.10e1.110.10efg0.240.01b0.160.02e Co-fermentedNDND0.390.01cd1.440.10b2.060.21a3.330.10b1.800.14a0.290.01a0.040.01fgh Candidarailenensis S18Single0.090.01a0.030.01ab0.360.01cd0.930.07hij1.760.18a2.920.12d1.090.12efgh0.230.01bc0.080.01f Co-fermentedNDND0.370.01cd1.120.04de1.940.16a3.150.08bcd1.540.11b0.220.02bcd0.030.01gh Metschnikowia pulcherrimaS36Single0.040.01bND0.300.01d0.880.03ij2.010.20a3.140.13bcd0.960.09gh0.220.01bcd0.080.01f Co-fermentedNDND0.370.02cd1.100.03def1.830.11a2.890.11d1.140.09efg0.210.01bcd0.010.01h Differentletterswithinthesamecolumnindicateasigniﬁcantdifference(P<0.05).ND,notdetected. Table2.ConcentrationofvolatilehigheralcoholsinCampbellEarlywinesfermentedwithindividualnon-Saccharomycesyeastsandco-fermentedwithindividualnon-SaccharomycesyeaststogetherwithS.cerevisiae. Concentrationofvolatilehigheralcohols(mg/L) InoculatedyeastInoculation strategy1-PropanolIso-butanolIsoamylalcohol1-Hexanol1-Heptanol2-Ethyl- 1-hexanol1-Octanol1-Nonanol SaccharomycescerevisiaeW3Single2.200.30e45.544.35fghi515.3234.90ab18.561.76gh2.930.35def0.580.05f1.630.14cd23.892.52ab Wickerhamomycesanomalus JK04Single3.870.30e38.713.41ghij265.0128.64g23.252.15cdefg1.420.21hi1.740.15d0.770.05g8.400.73j Co-fermented2.290.20e50.865.39efgh458.0840.04bc18.652.04gh3.010.27def0.620.05f2.680.26a19.301.62cde TorulasporadelbrueckiiJK08Single22.842.00c57.185.22cdef384.5133.77cde27.892.45bcd1.050.11i2.210.19b0.710.06g10.180.87ij Co-fermented2.260.24e37.303.38hij335.6130.89efg17.941.69gh1.750.21gh0.530.06f2.400.18a16.151.40efg StarmerellabacillarisMR35Single3.220.39e103.9711.17a163.0613.07h43.473.98a0.420.07j2.850.25a0.770.06g7.210.75j Co-fermented2.780.22e56.035.85cdef273.5923.92fg25.142.83bcdef1.830.17gh1.590.14d1.800.22bc14.931.53fg CandidaquercitrusaP6Single5.130.42e34.143.14ij360.2837.82def22.852.20defg2.690.32ef2.410.23b1.230.10e9.890.95ij Co-fermented2.200.29e44.884.14fghi433.9541.54bcd15.961.32h2.740.25ef0.580.07f2.590.27a18.291.67cdef PichiakluyveriP11Single11.070.99d47.373.53fghi353.9932.39def17.881.63ghND1.000.10e0.850.08fg10.071.04ij Co-fermented2.150.19e53.025.07defg481.5143.54ab20.471.96fgh3.530.31d0.660.05f2.390.21a18.731.94cde HanseniasporavineaeS7Single4.580.49e26.111.61j377.8132.32cde27.972.39bcd1.450.13hi1.990.19c1.340.15de25.112.44a Co-fermented2.790.31e42.854.49fghi362.0835.51def21.692.09efgh2.380.21efg0.540.05f1.370.15de21.131.89bcd H.uvarumS8Single25.750.22b66.126.15cd337.8930.43efg29.132.55bc4.840.42bc1.610.10d1.150.13ef13.741.31gh Co-fermented2.740.24e65.766.51cd506.0842.96ab20.372.17fgh5.280.47ab0.720.07f0.730.09g17.531.62def CandidarailenensisS18Single46.574.95a91.367.73b502.5749.26ab30.722.84b5.750.49a1.620.13d1.290.10e11.701.25hi Co-fermented2.330.23e69.486.35c560.8251.58a23.881.92cdefg4.510.43c1.050.11e2.590.12a21.481.69bc Metschnikowiapulcherrima S36Single4.450.43e86.818.48b481.1240.45ab27.063.42bcde3.090.35de1.570.12d1.110.07ef11.481.24hi Co-fermented1.950.18e63.825.92cde473.7542.59ab15.411.72h2.330.29fg0.480.04f1.990.18b16.441.36efg Differentletterswithinthesamecolumnindicateasigniﬁcantdifference(P<0.05).ND,notdetected.

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fermented wines, including the Control wine (inoculated withS. cerevisiae)], indicating that volatile higher alcohols in Campbell Early wines were inﬂuenced by S. cerevisiae because the Control wine (single-culture ofS. cerevisiae) was located in the centre of the other co-fermented wines. In addition, single-fermented wines were located on the right side of each co-fermented wine, depending on the species of non-Saccharomycesyeasts.

Sixteen volatile esters were detected in all wines (Table 3), with each ester showing different patterns depending on the strain and inoculation strategy, similar to volatile higher alcohols. Methyl acetate concentration of wines fermented by a single-culture of W. anomalus, P. kluyveri, H. uvarum and C. railenensis was signiﬁcantly higher than that of Control and co-fermented wines. In addition, the methyl acetate concentration of single- fermented wines by T. delbrueckiiand M. pulcherrima were slightly higher than that of co-fermented wines. Ethyl acetate concentration of wines fermented by a single-culture of W. anomalus, P. kluyveri, H. vineae, H. uvarum and M. pulcherrima and co-culture of H. vineae and H. uvarum were signiﬁcantly higher than that of the Control wine.

Isobutyl acetate and isoamyl acetate concentration of single- fermented wines of non-Saccharomycesyeasts was lower than that of the Control wine, and that of most co-fermented wines 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 ethyl butyrate, ethyl octanoate, ethyl decanoate, ethyl 9-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-fermented wines was lower than that in co-fermented wines (except for C. railenensis for ethyl butyrate; H. uvarum and C. railenensis for ethyl octanoate; C. railenensis and M. pulcherrimafor ethyl decanoate; M. pulcherrimafor ethyl 9-decenoate;C. railenensisandM. pulcherrimafor ethyl dodecanoate). Ethyl hexanoate concentration of most wines fermented by single-culture of non-Saccharomyces yeasts was signiﬁcantly lower than that in the Control, and wines fermented by single-culture ofC. railenensisandM. pulcherrima

showed similar ethyl hexanoate concentration when compared to that of the Control wine. The concentration of n-hexyl acetate of wines fermented by single-culture of non-Saccharomycesyeasts, except for P. kluyveri, was notably lower 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, was higher than that of the Control and co-fermented wines.

The concentration of methyl salicylate of all wines was not notably different; however, the concentration in wines fermented by single-culture of S. bacillaris andP. kluyveri was slightly higher than that in the Control wine. Ethyl phen- ylacetate concentration of all wines was mostly similar. The concentration of 2-phenylethyl acetate of wines fermented by single-culture ofP. kluyveriand single- and co-culture of H. vineae was signiﬁcantly higher; however, that of other wines was lower compared to the Control wine. Ethyl hexadecanoate concentration of co-fermented wines was higher than that in the Control wine, and that of wines fermented by single-culture ofH. uvarumandC. railenensiswas slightly higher than in the Control. The volatile ester compounds of all wines were subjected to PCA, and PC1 and PC2 represented 64.85 and 14.89% of the variance, respectively (Figure 6). Similar to the PCA plot of volatile higher alcohol compounds, volatile ester compounds formed two clusters, such as single-fermented wines (not inoculated with S. cerevisiae) and co-fermented wines (inoculated with S. cerevisiae). In addition, single-fermented wines were located on the left and upper side of each co-fermented wine, depending on the species of non-Saccharomycesyeast.

Hue and intensity of Campbell Early wine

The hue and intensity values of all the wines are listed in Table 4. Initial hue and intensity values of wines were 1.4060.023 and 3.4880.035, respectively. Hue values of all the wines fermented with a single species were higher than those of the Control and co-fermented wines, whereas the intensity values of most wines fermented with a single species were lower than those of the Control and co- fermented wines (except for C. quercitrusa). Hue and intensity values of wines fermented byW. anomalus,T. delbrueckii, S. bacillaris,H. vineaeand M. pulcherrimawere, respectively, signiﬁcantly higher and lower than those of the Control and co-fermented wines.

Sensory evaluation

Table 5 shows the sensory evaluation results of Campbell Early wines fermented by single-culture of non-Saccharomy- cesyeasts and by co-culture of those species andS. cerevisiae.

Some wines fermented with a single species that showed relatively high hue and low intensity values (W. anomalus, T. delbrueckii, S. bacillaris, H. vineae and M. pulcherrima) exhibited lower colour values compared with co-fermented wines. Furthermore, most wines fermented by a single- culture of non-Saccharomyces yeasts obtained higher scores for sweetness (except forC. quercitrusaandC. railenensis) and sourness (except for H. uvarum and M. pulcherrima) compared to those of co-fermented wines. As a result ofﬂavour and overall preference, wines fermented by single-culture of W. anomalusand H. uvarumand co-culture ofC. quercitrusa obtained higher scores compared to that of the Control wine. Additionally, wine fermented by co-culture of H. uvarumobtained a higherﬂavour score compared to that of the Control wine.

Figure 5. Proﬁling of volatile higher alcohols based on principal component analysis (PCA) and solid-phase microextraction (SPME)/GC/MS analysis of Campbell Early grape wine fermented by individual species of non-Saccharomycesyeasts ( ) and co-fermented by individual species of non-Saccharomyces yeasts together with S. cerevisiae W-3 ( ).

S. cerevisiae W-3 ( ); Wickerhamomyces anomalus JK04 ( , );

Torulaspora delbrueckii JK08 ( , ); Starmerella bacillaris MR35 ( , );

Candida quercitrusaP6 ( , ); Pichia kluyveriP11 ( , );Hanseniaspora vineaeS7 ( , );H. uvarumS8 ( , );Candida railenensisS18 ( , ); and Metschnikowia pulcherrimaS36 ( , ).