1. Introduction
The most common starter cultures employed in alcoholic fermentation are
Saccharomyces species. These species combine several desirable characteristics, such as efficiency in fermenting sugars and producing ethanol, synthesis of aromas, and high ethanol tolerance [
1]. However, in the early stages of wine fermentation, as well as in spontaneously fermented wines, several non-
Saccharomyces species also participate in the fermentation process. These species may have a positive impact on desirable organoleptic characteristics of the wine such as improved mouthfeel and more complex flavour profiles [
2].
Recently there has been an increasing interest in both winemaking and brewing towards non-Saccharomyces yeasts for their potential for aroma enhancement while maintaining the particular terroir of each region or for the development of craft beers.
In the case of wine, application studies with a selection of these so-called non-conventional yeasts indeed improve wine aroma; for example, the sequential fermentation of Ribolla Gialla grape juice employing commercial
Saccharomyces cerevisiae Lalvin T73 and
Zygosaccharomyces kombuchaensis CBS8849 or
Kazachstania gamospora CBS 10400 resulted in positive organoleptic properties and suitable fermentation dynamics, rapid sugar consumption and industrial strain compatibility [
3]. In addition, Jood et al. [
4] showed that mixed-culture fermentations involving
S. cerevisiae and different
Kazachstania species have very distinct aroma profiles, whereas Varela et al. [
5] showed that wines fermented with a combination of
Metschnikowia pulcherrima and
S. uvarum were characterised by increased concentrations of 2-phenylethanol and 2-phenylethyl acetate, both associated with positive sensory attributes. In simultaneous and sequential inoculations of selected
Rhodotorula mucilaginosa and
S.cerevisiae strains, both the variety and levels of fermentative aromacompounds were improved, especially that of (Z)-3-hexene-1-ol, nerol oxide, certain acetates and ethyl esters. These changes are associated with an improved perception of citrus, sweet fruit, acid fruit, berry, and floral aroma [
6].
In a similar approach, the use of non-
Saccharomyces yeasts in brewing has been explored [
7].
Torulaspora delbrueckii, for example, was studied in single-strain fermentations for the production of low-alcohol beer and for enhancing beer flavour profiles. Some strains fermented all the wort sugars, whereas others did not, but all strains tested displayed improved flavour-forming properties [
8]. In another study, the use of pure cultures of
T. delbrueckii and mixed cultures of
T. delbrueckii and
S. cerevisiae for brewing resulted in a well noticeable change in flavour profile due to
T. delbrueckii. Distinctive sensory profiles with pure cultures of
T. Delbrueckii were characterised by ‘fruit/citric’ notes and ‘full-bodied’ attributes whereas some mixed cultures enhanced the ‘fruity/ester’ and ‘hop’ aromas. At the same time, these beers showed enhanced clarity and a more persistent and compact foam [
2]. Another approach involving non-
Saccharomyces yeasts to produce low-alcohol beers was carried out by van Rijswijck et al. [
9]. These authors found that the non-conventional yeasts
Cyberlindnera fabianii and
Pichia kudriavzevii both in pure and mixed cultures with
S. cerevisiae yielded beer with lower ethanol content and higher levels of esters.
Brettanomyces custersii, isolated from Lambic beer, showed higher β-glucosidase activity than
S. cerevisiae, thus a higher potential for aroma release from precursors. Fermentation with pure cultures of a
Brettanomyces custersii strain or in a co-culture with a
S. cerevisiae resulted in enhanced release of volatiles from hop glycosides [
2]. In a more recent study, Holt et al. [
10] screened 17 non-conventional yeast species in sequential brewing fermentation, followed by inoculation of
S. cerevisiae. In these fermentations, it was possible to enhance banana flavour (isoamyl acetate) by
Pichia kluyverii, spicy notes (phenolic compounds) by
Brettanomyces species and clove-like aroma (4-vinylguaiacol) by
T. delbrueckii.Furthermore, Gutiérrez et al. [
11] explored the potential of 99 non-
Saccharomyces yeasts to produce pleasant fruity aromas in three industrial media (beer, wine and cider) through an olfactory assay. Of these, 21 yeasts were further evaluated for their aroma profile and fermentation capacity using wort, grape and apple juice. These authors reached the conclusion that the choice of yeast has more impact than the medium composition. The yeasts with the best opportunities to tune beer flavour belong to the species
Galactomyces geotrichum,
Kazachstania zonata,
Kluyveromyces lactis,
Lindnera meyerae,
Pichia kluyveri,
Starmera caribaea,
Yarrowia lipolytica and
Saccharomycodes ludwigii. Finally, Ravasio et al. [
12] explored the potential of 60 non-conventional yeasts to improve beer aroma and found that the species
Wickerhamomyces anomalus was a good candidate to combine with lager yeast to increase fruity aroma.
In this paper, we analyse the fermentation performance and the aroma potential in microscale of 10 non-Saccharomyces species employed as pure and mixed cultures co-inoculated in different ratios with one Saccharomyces strain on both wort and wine must, showing that variation in pitching rates is a valid option to control diversification as well as the additional value in analysing large amounts of culture combinations using microwine and microbeer. In addition, a comparison between microscale and labscale was carried out to analyse the correspondence in fermentation performance and aroma profiles.
2. Materials and Methods
2.1. Strains
Ten non-
Saccharomyces strains were included in this study and are listed in
Table 1 (Westerdijk Fungal Biodiversity Institute-CBS-KNAW, Utrecht, The Netherlands). In addition, two commercial
Saccharomyces species were included as references:
S.cerevisiae Lalvin T73 (Lallemand) in wine and
S. Pastorianus WS34/70 (Fermentis, Lesaffre) in beer.
2.2. Microscale Fermentations
Strains were pre-cultured overnight in GPY medium at 30 °C. After centrifugation (1000× g, 5 min), cells were resuspended in sterile Syrah must (Sofralab, Magenta, France) or enzymatically treated glucose wort (14.5°P) with no hops (Carlsberg, Copenhagen, Denmark). Wine must contained 250 g/L of sugars and was sterilized overnight using 1 mL/L dimethyl dicarbonate (Sigma-Aldrich, St. Louis, MO, USA) whereas wort was autoclaved.
Fermentations were carried out in triplicate employing 24-well microplates (MP), where each well contained 5 mL of must or wort. The MP were covered with a breathable seal (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 25 °C for 10 days in anaerobic jars. The strains were inoculated in MP as pure cultures (OD600 nm of 0.2) or mixed cultures in combination with S. cerevisiae Lalvin T73 (microwine) or WS34/70 (microbeer) in proportion 1:1 (each strain OD600 nm of 0.1). In addition, extra microbeer experiments were carried out employing OD600 nm of 0.2 of non-Saccharomyces yeasts and OD600 nm of 0.04 and 0.01 of WS34/70 (5:1 and 20:1 proportions, respectively). These extra experiments were done due to cultures mixed in 1:1 ratio resulted in microbeers similar as those produced by pure S. cerevisiae culture, indicating the outcompeting growth rate of the commercial strain.
2.3. Residual Sugars and Ethanol Analysis by High-Performance Liquid Chromatography (HPLC)
Mono/di-saccharides (glucose, fructose, maltose, sucrose, maltotriose) and ethanol were determined by HPLC (Thermo Fisher Scientific, USA) equipped with a refractive index detector Shodex RI 101. Prior to analyses, proteins and fat in the samples were precipitated by Grimbely Biggs reagents and removed after centrifugation.
The HPLC system was equipped with a Sil-10 AD vp injector, a LC-10ATvp pump and a CTO-6A oven. Sample volume of 25 µL was injected onto the mixed bed guard AG50W-X4 400 mesh (20 × 9 mm, packed with H+, Bio-Rad, Hercules, CA, USA) and AG3-X4A, 200–400 mesh (Bio-Rad, Hercules, CA, USA). The column (Aminex HPX-87P, 300 × 7.8 mm; BioRad, Hercules, CA, USA), was held at 80 °C. Mono/disaccharides and ethanol were eluted isocratically with miliQ water, flowing at a rate of 0.4 mL/min. The autosampler temperature was 10 °C. The detection was carried out by a refractive index detector ERC-7510 (Erma, Japan). Data analysis was conducted with Chromeleon software version 6.60 (Thermo Fisher Scientific, Waltham, MA, USA). External standards (97%–99% purity) for the indicated mono/di-saccharides and ethanol were obtained by Sigma-Aldrich (St. Louis, MO, USA) and a 4-point calibration curve was constructed.
2.4. Aroma Analysis by GC-MS
Aroma compounds, higher alcohols, esters and vicinal diketones (
Table 2) were extracted by headspace solid-phase microextraction (HS-SPME) employing a grey fibre DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) and determined by GC-MS. The fibre was exposed to the headspace of the samples during 15 min at 60 °C and desorbed during 2 min in the GC injector at 250 °C. The injection device was a Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland). The injected compounds were refocused at the beginning of the GC column by cryo-trapping at −110 °C (ThermoQuest Ce Instruments, Wigan, UK). Subsequently, the cryo-trap was heated to 200 °C at a rate of 50 °C/s.GC–MS analyses were carried out with a Finnigan Trace GC/MS (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a 30 m × 0.25 µm VF-wax ms, df = 0.5 µm, capillary column (Varian, Palo Alto, CA, USA). The chromatographic oven was initially set at 40 °C for 1 min, then raised to 250 °C at 20 °C/min and then kept at 250 °C for 3 min. The carrier gas Helium was kept at a constant flow of 1.5 mL/min. The total runtime was 14.5 min. Mass spectral data was collected over a range of m/z 35–250 in full scan mode (scan time 0.25 s). Quantification of the compounds was done using calibration curves employing external standards of each aroma compound purchased from Sigma-Aldrich (St. Louis, MO, USA). Data analysis was performed by the Xcalibur 2.1 software (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
2.5. Comparison of Microscale to Labscale
S. cerevisiae NIZO 900 was pre-cultured overnight at 30 °C in GPY (2% glucose, ScharlauChemie, Spain; 1% bacteriological peptone, Oxoid, UK; 0.5% yeast extract, Oxoid, UK). This strain was selected due to its good performance in fermenting alcoholic beverages. Afterwards, yeast cultures were centrifuged at 1000× g for 5 min, the medium was decanted, and the cells were subsequently re-suspended in triplicate in either 200 mL (bottles-lab scale) or 5 mL (microplates, MTPs-microscale) of Syrah must (Sofralab, Magenta, France) at a final OD600 nm of 0.1. Priortoinoculation, wine must was sterilized overnight with 1 mL/L dimethyl dicarbonate (Sigma-Aldrich, St. Louis, MO, USA). Samples were taken after 1, 3, 7 and 10 days of fermentation, centrifuged (4 °C, 1000× g, 5 min) and frozen immediately at −20 °C, except for OD measurements.
2.5.1. Biomass Analysis
Optical density measurement at 600 nm (OD600 nm) was carried out in a UV/visible spectrophotometer (Ultra-spec 2000, Amersham Pharmacia Biotech, Little Chalfont, UK). To correct for non-linearity of the assay, samples were pre-diluted in medium to obtain reads between 0.2 and 0.4.
2.5.2. Fermentation Kinetics and Aroma Profiles
Residual sugars and ethanol in the resulting fermentations were analysed by HPLC as described in
Section 2.3.
Aroma profiles were determined by headspace solid-phase dynamic extraction (HS-SPDE) in combination with GC-MS. The HS-SPDE extraction was carried out using a 2.5 mL HS-syringe with a polydimethylsiloxane active charcoal coated needle (PDMS/AC). After the equilibration of the samples at 60 °C for 15 min, 1 mL of the headspace was injected into the GC column (1 min at 250 °C in splitless mode). GC and MS conditions were the same as described in
Section 2.4.
2.6. Statistical Analysis
Multifactorial and one-way ANOVA were performed at 95% of confidence level (p value ≤ 0.05) with the results of aroma production and fermentation kinetics in all the experiments. Average comparison was done using Fisher (microwine) or Tukey’s (microbeer) tests. The statistical analyses were carried out by XLSTAT (Microsoft, Redmond, WA, USA).