Selection of Saccharomyces cerevisiae Isolates from Helan Mountain in China for Wine Production

Abstract

S. cerevisiae strains were isolated and identified from vineyards and the spontaneous fermentation must at the eastern foot of Helan Mountain in China, and their oenological properties and fermentation abilities were analyzed. From the total of 199 S. cerevisiae strains isolated and identified, 14 isolates (F4-13, F5-7, F5-9, F5-12, F5-18, F5-19, F5-21, F6-8, F6-23, F9-23, SXY-4, HT-10, ZXY-17, MXY-19) exhibited excellent tolerance to sugar, SO₂, and ethanol. Among the isolates, the strain F4-13 exhibited the better oenological properties, with low H₂S production (+), suitable flocculation ability (58.74%), and reducing-L-malic acid ability (49.07%), and generated high contents of polyphenol, anthocyanin, tannin, terpenes, and higher alcohols, which contributed to the improvement of the red fruity and floral traits of the wines. The obtained results provide a strategy for the selection of indigenous S. cerevisiae for wine fermentation to produce high-quality wine with regional characteristics.

1. Introduction

Wine is the product of complex interaction between grape berries, microbiota on grape peels, and their surroundings, originating from the vineyard and present throughout the fermentation, aging, and packaging processes [1,2]. Yeast plays a vital role during wine fermentation. It converts sugar into alcohol and carbon dioxide and secretes various secondary metabolites (glycerin, higher alcohols, fatty acids, esters, etc.) to modify the organoleptic properties of wine [3,4]. Currently, pure fermentation of the imported commercial S. cerevisiae strain has been a common biotechnological strategy for industrial wine production in China due to the excellent fermentative rate, controllable fermentative process, and stable quality compared to spontaneous fermentation [5]. However, the widespread use of imported commercial S. cerevisiae as a starter has reduced the biodiversity of indigenous yeasts and hindered the development of indigenous yeast with excellent fermentative properties, resulting in wines with reduced flavor complexity and regional and varietal characteristics [5,6].

Researchers and enologists have attempted methods of producing wines with complex flavor and outstanding organoleptic properties that cater to consumer preferences [7,8]. The co-fermentation of S. cerevisiae with non-Saccharomyces is an emerging strategy to improve the formation of glycerol, varietal and fermentative aroma compounds, and stable pigments to modify the organoleptic properties of specific wines [9,10]. The addition of exogenous compounds (e.g., enzymes, nitrogen sources, unsaturated fatty acids, amino acids, or polyphenols) during wine fermentation is considered an effective method for the organoleptic properties of wine in enhancing the volatile aroma compounds and modifying physicochemical compositions [11,12]. In addition, the inoculation of the selected indigenous S. cerevisiae strain into must is also an economical, practical, and rapid strategy to strengthen the regional and varietal features of wines, as they are better acclimated to the micro-area conditions of the wine grape production regions [13].

Sun, Hu, Zhang, Zhu and Tao [11] and Wang, et al. [14] indicated that the indigenous S. cerevisiae strains with native “terroir” traits showed fermentation variations and released distinguishable metabolites, which resulted in various flavor profiles of wines to retain its flavor complexity and its regional and varietal features. The eastern foot of the Helan Mountain (38°47′ N, 106°27′ E), located in Ningxia of China, is a desirable region for wine-grape cultivation (with a significant diurnal temperature difference and sufficient sunlight) and is one of the primary sources of wine grapes in China [15]. As it is a traditional wine-grape cultivation region, hundreds of microbiomes inhabit the region, including indigenous yeasts, which may be better suited to produce wines with regional features due to their long-term adaptive evolution [16,17]. The selected indigenous S. cerevisiae strains with excellent oenological property, fermentation ability, and volatile aroma compound production capacity can be used to ferment wines with flavor complexity and regional and varietal characteristics [18,19].

The study aims to isolate and select indigenous S. cerevisiae strains from the eastern foot of Helan Mountain that possess excellent oenological properties, fermentation ability, and volatile compound production capacity for local wine production to restore regional features. Firstly, indigenous S. cerevisiae strains were isolated from grape peels, leaves, and soils of nine vinicultural regions as well as spontaneous fermentation of Cabernet Sauvignon must from the eastern foot of Helan Mountain, and then identified by morphology on Wallerstein nutrient medium and 26S rDNA D1/D2 domain sequencing. Next, their oenological properties, including stress tolerance, flocculation ability, H₂S production, reducing-L-malic acid ability, and fermentability, were evaluated at laboratory scale. The selected indigenous S. cerevisiae strains from the eastern foot of Helan Mountain will provide potential starters for wine production with regional characteristics and a reference for selecting other yeast strains with specific regional features as potential starters.

2. Materials and Methods

2.1. Sampling and Yeast Isolation

Grape berries, leaves, and soils of nine vineyards from the eastern foot of Helan Mountain in Ningxia of China (Figure 1A), and spontaneous fermentation wine of Cabernet Sauvignon were sampled as yeast-isolated sources. Specifically, soils were collected from five positions 5~10 cm beneath vineyard soil surfaces, and fresh leaves, dried leaves, ripe grapes, and overripe grapes were sampled from vineyards with sterile bags and stored at 4 °C. Additionally, spontaneous fermentation with 1 ton of Cabernet Sauvignon must (247.22 g/L sugar, 4.00 g/L total acids, pH 5.10) harvested from the Helan Mountain vineyard in 2016 was performed at 28 °C, and 1 mL of wine was taken daily during wine fermentation.

The aseptic flask containing 10 g sample and 90 mL sterile normal saline was shaken at 160 rpm for 30 min at room temperature (approx. 23 °C) to elute microorganisms. Subsequently, 0.1 mL serially diluted samples were spread onto YPD agar plates supplemented with 100 mg/L chloramphenicol to inhibit bacterial growth. After inoculation at 28 °C for 2 d, 20~30 colonies from each plate were randomly chosen for purification. All colonies were preserved on YPD slants at 4 °C for the following experiments.

2.2. Grapes, Yeasts and Media

Cabernet Sauvignon containing 239.92 g/L sugar, 6.75 g/L total acids (expressed as tartaric acid) and pH 3.96, normally harvested from the Helan Mountain region vineyard in 2017, located in Ningxia, China. The indigenous S. cerevisiae strains were isolated from grape peels, leaves, soils, and spontaneous fermentation must of Cabernet Sauvignon in 2016 (Figure 1B). S. cerevisiae F33 (FSC) from Laffort Ltd. (Actiflore Cervisiae, Laffort Co., Bordeaux, France) was used as a reference strain.

Yeast extract peptone dextrose medium (YPD, 10.00 g/L yeast extract, 20.00 g/L peptone, and 20.00 g/L dextrose) was employed to enrich culture yeast strains.

(1)

Wallerstein laboratory nutrient agar medium (WL, 50.00 g/L dextrose, 0.125 g/L MgSO₄, 5.00 g/L tryptone, 0.022 g/L bromocresol green, 4.00 g/L yeast extract, 0.0025 g/L FeCl₃, 0.55 g/L KH₂PO₄, 0.0025 g/L MnSO₄, 0.425 g/L KCl, 15.00 g/L agar, and 0.125 g/L CaCl₂, pH 5.50) was used to differentiate yeast species according to the colony morphology and the yeast color.

(2)

Lysine agar medium (LYS, 10.00 g/L dextrose, 5.00 g/L lysine, 0.10 g/L KH₂PO₄, 0.10 g/L MgSO₄, 0.10 g/L complex vitamin, and 15.00 g/L agar, pH 4.80) was used to differentiate S. cerevisiae and non-Saccharomyces strains.

(3)

Synthetic grape juice (SGJ, 100.00 g/L dextrose, 100.00 g/L fructose, 1.50 g/L yeast extract, 0.30 g/L citric acid, 5.00 g/L tartaric acid, 5.00 g/L malic acid, 2.00 g/L ammonium sulfate, 5.00 g/L potassium dihydrogen phosphate, 0.50 g/L magnesium sulfate, 0.20 g/L sodium chloride, and 0.05 g/L manganese sulfate, pH 3.20) was used as an alternative to the must for its precise physicochemical composition.

(4)

Bismuth sulfite agar medium (BSA, polypeptone 10.00 g/L, beef extract 5.00 g/L, dextrose 5.00 g/L, bismuth sulfite 8.00 g/L, FeSO₄ 0.30 g/L, Na₂HPO₄ 4.00 g/L, brilliant green 0.025 g/L, and agar 15.00 g/L, pH 7.70 ± 0.20) was used to test the hydrogen sulfide (H₂S) production of S. cerevisiae strains.

(5)

Yeast exact, L-malic acid peptone medium (YMP, 40.00 g/L L-malic acid, 10.00 g/L yeast extract, 20.00 g/L peptone, and 50 μL of 0.003 g/L bromocresol green solution) was used to evaluate the reducing-L-malic acid ability of S. cerevisiae strains.

2.3. Morphological and Molecular Identification of S. cerevisiae

The isolated strains were dotted on WL medium in duplicate and incubated at 28 °C for 5 d, and the colony morphologies were observed. When the front side of the colony was cream with little green, raised and opaque, and the back side was white or with little green, it was initially considered as S. cerevisiae. Then, the molecular identification of the isolated strains was performed by the analysis of 26S rDNA D1/D2 domain sequencing. Specifically, genomic DNA in yeast was extracted according to Sun, et al. [20]. The 26S rDNA D1/D2 domain was amplified with primers NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′) [21]. PCR amplification was performed in a 50 μL volume containing 1 μL DNA template (250 ng/μL), 4.0 μL dNTP (2.5 mmol/L each), 5.0 μL 10×PCR buffer solution (Mg²⁺ plus), 25 μL premix Taq polymerase (TAKARA, Beijing, China), and 3 μL each primer (10 μM). The PCR reaction was performed according to the following conditions: denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min and extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis on a 1% agarose gel, then purified and sequenced by Sangon Biotech (Shanghai, China) Co., Ltd. The 26S rDNA D1/D2 sequence amplified from each yeast strain was compared to the sequence stored in Genbank^® (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 10 March 2017) by using BLAST software [22]. Generally, the difference in the 26S rDNA D1/D2 domain sequence of the same species is within 1%.

2.4. Oenological Properties of S. cerevisiae

2.4.1. Tolerance to Sugar, SO₂, and Ethanol Analysis

The tolerances of the isolated yeast strains and FSC to sugar, SO₂, and ethanol were analyzed in a 96-well plate. Specifically, each well contained 200 μL SGJ with different concentrations of sugar (250 g/L), SO₂ (100 mg/L), ethanol (12% v/v), and 10⁶ CFU/mL yeast cells (prepared by culturing in YPD at 28 °C and 120 rpm for 24 h). After inoculation, plates were incubated at 28 °C for 48 h and microshocked until turbid, and then the absorbance was measured at 600 nm using a Multifunctional microplate reader (Synergy HTX, BioTek, Emeryville, CA, USA). Relative growth rate (RG, %) was used to evaluate the tolerance abilities of isolated yeast strains to sugar and the absorbance of FSC at 600 nm (AbsS) at the same sugar concentration as a reference, whereas inhibition rate (IR, %) was used to evaluate the tolerance abilities of isolated yeast strains to SO₂ and ethanol. RG (%) and IR (%) were calculated with the following formula:

$$\mathrm{RG} (\%)=\frac{AbsE}{AbsS}\times 100\%; \mathrm{IR} (\%)=(1-\frac{AbsE}{AbsE0 })\times 100\%$$

where AbsE is the absorbance of each sample at 600 nm in SGJ with different concentrations of sugar, SO₂, or ethanol; AbsS is the absorbance of FSC at 600 nm at different concentrations of sugar, and AbsE0 is the absorbance of each sample at 600 nm in the SGJ without SO₂ and ethanol.

2.4.2. Hydrogen Sulfide Production

H₂S productions of yeast strains were determined with a bismuth sulfite agar medium (BSA, Angel, Yichang, China) [23]. Activated yeast cells were collected by centrifugation (6000 rpm, 5 min) and washed with sterile saline 3 times. Then, 10 μL yeast solution (10⁷ CFU/mL) diluted with sterile saline was added dropwise on the surface of bismuth sulfite agar medium plate and incubated at 28 °C for 6 d, and the halo color surrounding the yeast colonies was measured. Generally, the strain resulted in higher H₂S production as the color of the halos deepened. The relationships among the symbol, H₂S production, and halo color were indicated as follows: − no (no change); + little (white); ++ low (light brown); +++ medium (green); ++++ high (dark green).

2.4.3. Flocculation Ability

The flocculation abilities of yeast strains were analyzed by the modified Helm method [24]. Specifically, yeast suspension (10⁸ CFU/mL) was centrifuged at 8000 rpm for 5 min, then the supernatant was discarded and labeled A and B. The sediment in A tube was mixed with an appropriate content of EDTA buffer (0.5 mol/L, pH 7.0) to obtain 10⁷ CFU/mL solution, and then its absorbance at 600 nm (Abs0) was measured. The sediment in B tube was washed with 0.51 g/L of calcium sulfate solution, then was mixed with 1 mL buffer (pH 4.5) containing 0.51 g/L of calcium sulfate, 6.80 g/L of sodium acetate, and 4.05 g/L of glacial acetic acid, and stood at room temperature for 15 min. Next, 100 μL of the supernatant was added into 900 μL water and vortexed, and the absorbance at 600 nm (Abs15) was measured. Flocculation abilities of yeast strains were evaluated by the following formula.

$$\mathrm{Flocculation} \mathrm{ability} (\%)=(1- \frac{Abs15}{Abs0})\times 100\%$$

2.4.4. Reducing-L-Malic Acid Ability

The reducing-L-malic acid abilities of yeast strains were assessed using YMP medium with bromocresol green. YMP medium with 10⁷ CFU/mL yeast cells was cultured at 28 °C for 6 d, and the color of the broth was observed. The color of the broth gradually varied from brown to light yellow or dark green as the amount of residual L-malic acid decreased. Residual L-malic acid in broth was detected by high-performance liquid chromatography (HPLC, Shimadzu Corporation, Kyoto, Japan) with Kromasil C₁₈ column (250 mm × 4.6 mm, 5 μm), the UV detector (SPD-40/SPD-40V) with 210 nm detection wavelength. A quantity of 10 μL of fermentation supernatant was separated with mobile phase composed of 1% acetonitrile and 0.02 mol/L potassium dihydrogen phosphate solution (pH 2.80) at a ratio of 1:9 and a flow rate of 0.50 mL/min at 30 °C. The abilities of yeast strains to reduce L-malic acid were evaluated by the following formula.

$$\mathrm{Reducing}-\mathrm{L}-\mathrm{malic} \mathrm{acid} \mathrm{ability} (\%)=(1– \frac{\mathrm{Residual} \mathrm{L}-\mathrm{malic} \mathrm{acid} \mathrm{content}}{\mathrm{Total} \mathrm{L}-\mathrm{malic} \mathrm{acid} \mathrm{content}})\times 100\%$$

2.5. Laboratory-Scale Production of Wines

The laboratory-scale vinification was conducted in 1 L sterile flasks containing 800 mL crushed Cabernet Sauvignon (pasteurized at 80 °C for 30 min) with the additional 60 mg/L of SO₂, and then macerated at 4 °C for 12 h before inoculation. After the treated must was restored to 25 °C, it was inoculated with the selected S. cerevisiae or the commercial S. cerevisiae F33 at 10⁶ CFU/mL, and fermented at 25 °C (±1 °C), respectively. The biomass of yeasts was monitored using the diluted coating plate method, and the residual sugar of yeasts was analyzed using the DNS colorimetry method every day during wine fermentation. The final wines were added with 50 mg/L SO₂. Wines fermented by 14 S. cerevisiae isolated strains and FSC were named as SCw and FSCw, respectively.

2.5.1. Physicochemical Composition Analysis of Wines

Ethanol, total acids (expressed as tartaric acid), and volatile acids (expressed as acetic acid) in wines were determined according to International Organization of Vine and Wine (OIV, Paris, France, 2016). Polyphenols, anthocyanins, and tannins in wines were evaluated by the folin-phenol colorimetry [25], pH differential [26], and colorimetry methods [27], respectively. The pH value of wines was determined by a pH meter (Mettler-Toledo, Shanghai, China).

2.5.2. Volatile Compounds Analysis of Wines

Volatile compounds in wines were detected using gas chromatography-mass spectrometry (GC–MS) coupled with headspace solid-phase microextraction (HS-SPME) adapted from Wang, et al. [28]. Volatile compounds were extracted using a 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA). A 20 mL gas-tight vial containing 2 g NaCl, 8 mL wine, and 10 μL internal standard (cyclohexanone) was placed in a 40 °C water bath with stirring at 600 rpm for 15 min, extracted for 30 min, and then desorbed in the GC injection port at 250 °C for 5 min using a Shimadzu TQ8040 GC–MS (Shimadzu Corporation, Kyoto, Japan) and a DB-WAX column (30 m × 0.25 mm × 0.25 μm, Agilent J & W, Santa Clara, CA, USA). The GC program was set as follows: the carrier gas was ultrapure helium (99.999%) with a flow rate of 1.00 mL/min; the temperature was increased from 40 °C to 130 °C at 3 °C/min, then ramped to 250 °C at 4 °C/min and maintained for 5 min. The MS was operated in electron impact ionization mode at 70 eV, and ion source temperature was 250 °C. Volatile compounds were identified by comparing their MS fragmentation pattern with those in database NIST 17 Volatile compound concentrations were quantitated by the following formula.

$$\mathrm{Compound} \mathrm{concentration} (\mathrm{mg}/\mathrm{L})=\frac{\mathrm{GC} \mathrm{peak} \mathrm{areas} \mathrm{of} \mathrm{the} \mathrm{compond} \times \mathrm{Quantity} \mathrm{of} \mathrm{internal} \mathrm{standard} \left(\mathrm{mg}\right)}{\mathrm{GC} \mathrm{peak} \mathrm{area} \mathrm{of} \mathrm{the} \mathrm{internal} \mathrm{standard} \times \mathrm{Volume} \mathrm{of} \mathrm{the} \mathrm{sample} \left(\mathrm{L}\right)}$$

2.5.3. Sensory Evaluation of Wines

Sensory evaluation of the wines was conducted by 17 well-trained panelists (8 males and 9 females) in a tasting room at 20 °C [11]. Wine samples of approximately 20 mL were poured into black INAO glass in triplicate. The wine aromas of wine were described using 4~6 terms, and the intensities of the aromas were scored using a five-point scale (1. very weak; 2. weak; 3. medium; 4. intense; 5. very intense). The radar charts of the aromatic characteristics of wines were plotted.

2.6. Statistical Analysis

All statistical data were analyzed by GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Sampling sites were drafted by Adobe Illustrator CS6 (Adobe Software Inc., Adobe, CA, USA). Significant differences (p < 0.05) among different S. cerevisiae strains were compared by Duncan’s multiple tests using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Hierarchical Cluster and Heatmap Diagram was used to describe the volatile compound profiles of different wine samples by OmicShare Tools (GENE DENOVO, Guangzhou, China) after Z-score standardization.

3. Results and Discussion

3.1. Identification of Isolated Yeasts

A total of 1066 yeast and yeast-like strains (hereby referred to as yeasts) were isolated from grape peels, leaves, soils, and spontaneous Cabernet Sauvignon must at the eastern foot of Helan Mountain in Ningxia, China (Figure 1A). Among these 1066 yeast strains, 199 S. cerevisiae strains were identified by the morphology characteristics on WL medium and partial 26S rDNA D1/D2 domain sequence (Tables S1 and S2). The number and isolation ratio of S. cerevisiae from different isolated sources were calculated, as shown in Figure 1B. Results indicated that spontaneous fermentation must (FM) was the best source for collecting S. cerevisiae, and 142 strains accounting for 71.36% of the total strains were isolated from FM, followed by grape leaves (34 strains, 17.09%), soils (22 strains, 11.05%) and grape peel (1 strain, 0.50%). Previous studies have shown that S. cerevisiae was abundant during spontaneous alcoholic fermentation, especially in the mid and late stages [13,29], but not easily found on the berries [30,31].

3.2. Oenological Properties of Yeasts

3.2.1. Tolerance Abilities of Yeasts to Sugar, SO₂, and Ethanol

The tolerance abilities analysis of 199 S. cerevisiae strains to sugar, SO₂, and ethanol showed that 196 strains had decent growth under 250 mg/L sugar (RG > 50%, Figure 2A), and 96 strains had decent growth under 100 mg/L SO₂ (IR > 50%, Figure 2B), while 15 strains exhibited decent growth under 12% (v/v) ethanol (IR > 50%, Figure 2C). Among them, S. cerevisiae F4-13, F5-7, F5-9, F5-12, F5-18, F5-19, F5-21, F6-8, F6-23, F9-23, SXY-4, HT-10, ZXY-17, and MXY-19 showed normal growth under 250 g/L sugar, 100 mg/L SO₂, and 12% (v/v) ethanol (Figure 2D and Table S3). Among 14 S. cerevisiae with good tolerance, 10 strains were isolated from the spontaneous fermentation of Cabernet Sauvignon, suggesting that S. cerevisiae from spontaneous fermentation had better stress resistance than those from other isolated sources.

3.2.2. Properties of H₂S Production, Flocculation, and Reducing-L-Malic Acid of Yeasts

Fourteen S. cerevisiae strains with good tolerance to sugar, SO₂, and ethanol were further selected by analyzing their oenological properties. The results in Figure 3A indicated that 14 strains presented lower H₂S production on BAS plate than FSC, and F4-13, F5-7, HT-10, and ZXY-17 barely produced H₂S. High content of H₂S presents “rotten eggs” odor and damages the organoleptic quality of wine, while low H₂S production favors the organoleptic quality of wine [32,33]. Therefore, low H₂S production is one of the evaluating or selecting criteria of that yeast strains to be used as potential starters to ferment musts [34,35].

As shown in Figure 3B, the 14 S. cerevisiae strains exhibited 22.17~97.16% flocculation abilities except for F5-9 and ZXY-17, and strains F9-23 (97.16%), F6-23 (92.70%), MXY-19 (89.37%), F5-19 (88.60%), HT-10 (80.58%), F5-18 (76.7%), and F4-13 (58.74%) displayed stronger flocculation abilities than FSC (54.28%). Traditionally, strong flocculation of yeast is in favor of the separation of cell from broth after fermentation [36].

Additionally, the reducing-L-malic acid abilities of 14 selected strains ranged from 11.70% to 72.50%, and HT-10 (72.50%), F6-8 (56.27%), MXY-19 (53.80%), ZXY-17 (53.13%), F4-13 (49.07%), and SXY-4 (44.50%) presented stronger reducing-L-malic acid ability (>44.50%) than FSC (39.60%) (Figure 3D). The results obtained from the color indicator of YMP was highly consistent with that from HPLC analysis (Figure 3C). A reasonable acidity is important to ensure high-quality wine, but high acidity, especially a high level of L-malic acid, will destroy the organoleptic properties of wine [37,38]. Therefore, the reducing-L-malic acid ability is considered a desirable property of yeast strains to achieve a balanced taste in wine [39].

3.3. Performance of the Selected Yeasts during Alcoholic Fermentation

3.3.1. Growth Kinetics and Sugar Consumptions

As shown in Figure 4, the 14 S. cerevisiae strains almost reached their maximum biomass (>10⁸ CFU/mL) between the 1st and 4th days of wine fermentation, and the biomass of F5-7 (6.70 × 10⁸ CFU/mL), F5-9 (5.25 × 10⁸ CFU/mL), and F5-18 (5.05 × 10⁸ CFU/mL) exceeded those of FSC (3.60 × 10⁸ CFU/mL) after one day of wine fermentation (Figure 4C,D,F). The fermentation periods of 14 S. cerevisiae isolated strains were 5–8 d, except for F4-13 (8 d), and those of 13 strains were shorter than that of FSC (8 d). Among them, the fermentation period of F5-7, F5-12, F5-18, and MXY-19 was 5 d (Figure 4C,E,F,O), that of F5-9, F5-19, F5-21, F6-23, and ZXY-17 was 6 d (Figure 4D,G,H,J,N), and that of F6-8, F9-23, SXY-4, and HT-10 was 7 d (Figure 4I,K,L,M). These results indicated that 14 S. cerevisiae strains displayed excellent growth and fermentation abilities.

3.3.2. Physicochemical Compositions of Wines

Table 1. Physicochemical compositions of wines fermented by the selected S. cerevisiae strains.

Wines	Residual Sugar	Ethanol	Total Acids A	Volatile Acids B	Polyphenols	Anthocyanin	Tannin	pH
	g/L	% (v/v)	g/L	g/L	g/L	mg/L	g/L
FSCw	3.74 ± 0.05 gh	12.65 ± 0.34 hi	4.97 ± 0.23 b	0.09 ± 0.00 cd	2.00 ± 0.01 b	97.13 ± 1.28 a	3.76 ± 0.30 a	3.23 ± 0.02 d
F4-13w	3.31 ± 0.09 cd	11.58 ± 0.34 bc	5.93 ± 0.23 e	0.09 ± 0.00 bc	2.65 ± 0.08 i	151.96 ± 1.45 d	4.55 ± 0.08 b	3.32 ± 0.02 f
F5-7w	2.13 ± 0.03 a	12.20 ± 0.08 ef	6.12 ± 0.05 e	0.09 ± 0.00 bc	2.50 ± 0.03 fgh	208.18 ± 1.28 g	4.73 ± 0.16 bc	3.32 ± 0.02 f
F5-9w	3.25 ± 0.20 cd	11.81 ± 0.32 cd	5.93 ± 0.23 e	0.07 ± 0.00 a	2.29 ± 0.03 cd	110.21 ± 3.64 b	5.65 ± 0.18 d	3.38 ± 0.02 gh
F5-12w	2.92 ± 0.04 b	12.24 ± 0.32 ef	6.09 ± 0.00 e	0.11 ± 0.00 e	2.52 ± 0.13 fgh	202.61 ± 4.75 g	4.72 ± 0.09 bc	3.27 ± 0.04 e
F5-18w	2.89 ± 0.43 b	12.09 ± 0.15 de	5.39 ± 0.09 c	0.08 ± 0.00 b	2.56 ± 0.12 hi	156.97 ± 2.21 de	5.35 ± 0.52 d	3.35 ± 0.01 fg
F5-19w	3.43 ± 0.07 cd	11.39 ± 0.08 ab	6.41 ± 0.00 f	0.13 ± 0.00 g	2.33 ± 0.03 de	154.46 ± 0.83 de	4.66 ± 0.21 bc	3.12 ± 0.02 b
F5-21w	3.53 ± 0.04 de	12.92 ± 0.19 i	5.23 ± 0.14 c	0.09 ± 0.00 cd	2.20 ± 0.03 c	158.08 ± 9.89 de	3.90 ± 0.25 a	3.34 ± 0.01 f
F6-8w	3.40 ± 0.18 cd	12.20 ± 0.11 gh	5.39 ± 0.09 c	0.09 ± 0.00 bc	2.44 ± 0.02 fg	224.32 ± 2.55 h	3.90 ± 0.05 a	3.06 ± 0.01 a
F6-23w	3.87 ± 0.13 h	12.65 ± 0.34 hi	6.12 ± 0.05 e	0.10 ± 0.00 d	2.55 ± 0.04 ghi	249.09 ± 1.74 i	4.53 ± 0.26 b	3.17 ± 0.02 c
F9-23w	3.27 ± 0.13 cd	11.31 ± 0.27 ab	6.66 ± 0.09 g	0.09 ± 0.00 bc	2.84 ± 0.07 j	242.69 ± 13.26 i	4.50 ± 0.06 b	3.24 ± 0.04 de
SXY-4w	2.92 ± 0.04 b	12.09 ± 0.23 de	5.67 ± 0.32 d	0.15 ± 0.01 h	2.29 ± 0.04 cd	162.54 ± 2.68 e	4.98 ± 0.04 c	3.40 ± 0.02 i
HT-10w	3.61 ± 0.11 fgh	11.22 ± 0.32 a	6.34 ± 0.09 f	0.12 ± 0.00 f	1.89 ± 0.00 a	119.40 ± 2.50 c	3.88 ± 0.18 a	3.05 ± 0.01 a
ZXY-17w	3.57 ± 0.23 ef	12.12 ± 0.23 de	4.75 ± 0.09 a	0.19 ± 0.00 i	2.42 ± 0.03 ef	183.41 ± 3.16 f	4.98 ± 0.06 c	3.33 ± 0.02 f
MXY-19w	3.13 ± 0.04 bc	12.50 ± 0.06 efg	6.34 ± 0.36 f	0.12 ± 0.00 fg	2.20 ± 0.04 c	150.85 ± 0.48 d	4.37 ± 0.14 b	3.38 ± 0.02 gh

Notes: Values with different lowercase letters in the same row are significantly different according to the Duncan test (p < 0.05). A: as acetic acid; B: as tartaric acid.

shows that compared to FSCw, SCw had lower residual sugar content (2.13~3.57 g/L) except for HT-10w and F6-23w, lower ethanol content (11.22~12.50% v/v) except for F5-21w and F6-23w, lower pH value (3.82~4.06), higher total acids content (5.39~6.66 g/L) except for ZXY-17w, higher polyphenol content (2.20~2.65 g/L) except for HT-10w, higher anthocyanin content (110.21~249.09 mg/L), and higher tannin content (4.55~5.65 g/L). In addition, the volatile acids content (0.08~0.19 g/L) of all wines was well below 1.2 g/L. The results showed that these physicochemical compositions of the wines met the requirements of National Standard of the People’s Republic of China (GB/T 15038-2006, 2006). Interestingly, Qi, et al. [40] found that the wine of the Zhihuiyuanshi vineyard exhibited higher tannins and total phenols than the other three vineyards. Similarly, ZXY-17 obtained from the Zhihuiyuanshi vineyard in the study also showed relatively higher tannins (4.98 ± 0.06 g/L) and total phenols content (2.42 ± 0.03 g/L). In addition, the physicochemical indicators of the wines (pH: 3.05~3.40, alcohol: 11.31~12.65% (v/v), total phenolic content: 1.89~2.84 g/L) in our study were consistent with the same wine-producing region of China (pH: 3.59~3.77, alcohol: 12.10~13.40% (v/v), total phenolic content: 1.87~2.04 g/L).

3.3.3. Volatile Compounds of Wines

As shown in

Table 2. Analysis of volatile aroma compounds in wines fermented by the selected S. cerevisiae strains (mg/L; means ± SD).

Aroma Compounds	FSCw	F4-13w	F5-7w	F5-9w	F5-12w	F5-18w	F5-19w	F5-21w	F6-8w	F6-23w	F9-23w	SXY-4w	HT-10w	ZXY-17w	MXY-19w	Threshold *	Description	OVA
C6 alcohols	1.33 ± 0.08 a	1.04 ± 0.01 abc	0.68 ± 0.22 c	0.72 ± 0.01 abc	0.66 ± 0.06 bc	0.69 ± 0.40 abc	0.44 ± 0.23 c	0.64 ± 0.30 bc	0.89 ± 0.07 abc	0.60 ± 0.27 bc	0.42 ± 0.13 c	1.10 ± 0.02 ab	1.09 ± 0.09 ab	0.88 ± 0.05 abc	0.91 ± 0.46 abc
1-Hexanol	0.45 ± 0.03 def	0.51 ± 0.02 ef	0.39 ± 0.01 cd	nd	0.46 ± 0.01 def	0.27 ± 0.04 ab	0.19 ± 0.02 a	0.35 ± 0.04 bcd	0.47 ± 0.11 def	0.26 ± 0.02 ab	0.22 ± 0.02 a	0.29 ± 0.00 abc	0.57 ± 0.04 f	0.27 ± 0.04 ab	0.41 ± 0.03 de	8 A	Green, gross	<0.1
cis-2-Hexen-1-ol	0.88 ± 0.05 a	0.53 ± 0.03 abc	0.28 ± 0.21 bc	0.72 ± 0.01 abc	0.20 ± 0.07 c	0.43 ± 0.36 abc	0.25 ± 0.21 bc	0.28 ± 0.25 bc	0.42 ± 0.04 abc	0.34 ± 0.28 abc	0.20 ± 0.15 c	0.81 ± 0.02 ab	0.52 ± 0.06 abc	0.61 ± 0.01 abc	0.50 ± 0.43 abc	0.4 A	Green, cypress	0.1–1
Terpenes	0.56 ± 0.01 a	0.66 ± 0.04 a	0.33 ± 0.01 bcd	0.24 ± 0.12 b	0.01 ± 0.00 e	0.64 ± 0.18 a	0.29 ± 0.11 bcd	0.42 ± 0.07 d	0.39 ± 0.00 cd	0.04 ± 0.00 e	0.04 ± 0.01 e	0.28 ± 0.02 bc	0.27 ± 0.08 bc	0.03 ± 0.00 e	0.41 ± 0.01 d
Citronellol	0.09 ± 0.03 abc	0.14 ± 0.02 b	0.04 ± 0.00 ac	0.03 ± 0.01 a	0.01 ± 0.00 a	0.04 ± 0.00 ac	0.03 ± 0.02 a	0.03 ± 0.01 a	0.09 ± 0.00 abc	nd	nd	0.04 ± 0.02 ac	0.12 ± 0.09 bc	nd	0.06 ± 0.00 abc	0.1 A	Lemon green	0.1–1
Linalool	0.20 ± 0.00 a	0.20 ± 0.02 a	0.13 ± 0.00 c	0.09 ± 0.07 bcd	nd	0.12 ± 0.07 bc	0.12 ± 0.04 bc	0.04 ± 0.03 d	0.09 ± 0.01 bcd	0.04 ± 0.00 d	nd	0.07 ± 0.02 bcd	0.06 ± 0.00 bd	nd	0.08 ± 0.01 bcd	0.015 A	Floral, fruity, muscat	>1
Nerolidol	0.03 ± 0.00 a	0.08 ± 0.00 b	nd	nd	nd	nd	0.02 ± 0.02 a	nd	0.03 ± 0.02 a	nd	nd	nd	nd	nd	0.08 ± 0.02 b	0.7 A	Rose, apple, citrus	<0.1
cis-Geraniol	0.10 ± 0.01 a	0.11 ± 0.01 ab	0.08 ± 0.00 ab	0.05 ± 0.02 bc	nd	0.07 ± 0.02 abc	0.03 ± 0.01 c	nd	0.06 ± 0.01 bc	nd	nd	nd	nd	nd	nd	0.03 A	Tropical fruity, grass	>1
Geranyl acetone	0.09 ± 0.00 a	0.03 ± 0.01 a	0.04 ± 0.00 a	0.01 ± 0.00 a	nd	0.34 ± 0.30 b	nd	nd	0.06 ± 0.01 a	nd	0.04 ± 0.01 a	0.03 ± 0.00 a	0.03 ± 0.00 a	nd	0.08 ± 0.01 a	0.06 A	Floral	>1
Citronellol acetate	0.04 ± 0.00 ab	0.05 ± 0.01 bc	nd	0.03 ± 0.02 a	nd	0.03 ± 0.01 a	0.02 ± 0.01 a	0.04 ± 0.00 ab	0.04 ± 0.00 ab	nd	nd	0.06 ± 0.00 cd	0.05 ± 0.01 bc	0.03 ± 0.00 a	0.07 ± 0.01 d	0.25 A	Fresh, fruity, rose	0.1–1
Nerol acetate	nd	0.05 ± 0.01 ab	0.04 ± 0.00 ab	0.02 ± 0.01 a	nd	0.04 ± 0.02 ab	0.06 ± 0.00 bc	0.31 ± 0.04 d	0.03 ± 0.01 ab	nd	nd	0.08 ± 0.01 c	nd	nd	0.05 ± 0.00 ab	3 A	Petroleum, floral	<0.1
Fermentative aroma	1147.85 ± 36.10 d	994.90 ± 39.98 cd	664.89 ± 113.19 ab	550.22 ± 313.48 a	730.68 ± 88.83 abc	501.42 ± 201.99 a	484.50 ± 118.34 a	955.18 ± 285.05 bcd	493.74 ± 91.45 a	659.31 ± 116.11 ab	503.41 ± 87.35 a	887.96 ± 62.95 bcd	690.92 ± 35.70 ab	716.14 ± 140.83 abc	1044.55 ± 317.72 d
Higher alcohols	273.39 ± 1.86 abc	373.92 ± 9.30 d	286.57 ± 9.48 ac	157.88 ± 83.94 e	424.30 ± 15.74 d	171.99 ± 32.41 e	288.55 ± 54.81 ac	424.05 ± 61.17 d	214.03 ± 29.71 be	294.55 ± 16.55 a	219.85 ± 43.71 bce	263.5 ± 26.53 abc	370.82 ± 6.01 d	218.9 ± 37.86 bce	296.35 ± 13.32 a
1-Butanol	0.49 ± 0.01 a	0.24 ± 0.02 b	0.85 ± 0.03 cd	0.46 ± 0.26 a	0.57 ± 0.02 a	0.92 ± 0.10 cd	0.70 ± 0.14 ac	1.50 ± 0.21 e	nd	1.04 ± 0.12 d	0.58 ± 0.12 a	1.49 ± 0.19 e	2.59 ± 0.13 f	1.05 ± 0.16 d	1.51 ± 0.01 e	150 A	Medicinal, alcohol	<0.1
1-Decanol	0.12 ± 0.01 a	0.11 ± 0.01 ab	0.09 ± 0.02 bc	0.05 ± 0.02 ef	0.05 ± 0.01 efg	0.07 ± 0.03 cde	nd	0.05 ± 0.00 efg	0.09 ± 0.00 b	0.07 ± 0.01 de	0.02 ± 0.00 g	0.05 ± 0.00 ef	0.04 ± 0.01 fg	nd	0.08 ± 0.00 bcd	0.4 A	Orange, flowery	0.1–1
1-Nonanol	0.46 ± 0.04 a	0.34 ± 0.02 abc	0.18 ± 0.09 de	0.26 ± 0.14 be	0.04 ± 0.00 a	0.37 ± 0.08 abc	0.05 ± 0.01 ab	0.10 ± 0.05 ab	nd	nd	0.11 ± 0.00 ab	0.43 ± 0.12 ac	0.25 ± 0.06 be	0.14 ± 0.08 def	0.32 ± 0.10 bc	0.6 A	Green	0.1–1
1-Octanol	0.48 ± 0.01 abc	0.61 ± 0.02 b	0.50 ± 0.10 abc	0.37 ± 0.20 acde	nd	0.52 ± 0.21 ab	0.26 ± 0.05 de	0.34 ± 0.03 cde	0.35 ± 0.07 cde	0.30 ± 0.04 de	0.21 ± 0.03 d	0.39 ± 0.08 ace	0.33 ± 0.01 cde	0.23 ± 0.04 de	0.35 ± 0.03 cde	0.9 A	Intense citrus, rose	0.1–1
Isobutanol	7.33 ± 0.06 a	13.15 ± 0.56 b	7.02 ± 0.25 ac	3.42 ± 1.79 d	11.84 ± 1.56 be	3.82 ± 0.48 dg	10.10 ± 2.90 ef	8.08 ± 1.28 af	35.30 ± 2.30 h	10.72 ± 0.80 e	7.91 ± 1.55 af	5.91 ± 1.01 acg	7.25 ± 0.27 a	4.65 ± 0.80 cdg	5.92 ± 0.17 acg	40 A	Alcohol, nail polish	0.1–1
Isoamylol	173.94 ± 0.74 ab	242.62 ± 15.47 cd	195.45 ± 7.10 abd	112.15 ± 59.51 e	308.35 ± 6.20 g	117.89 ± 22.43 e	196.23 ± 36.84 ad	295.50 ± 42.05 fg	176.94 ± 27.40 ab	187.96 ± 9.52 ab	145.6 ± 29.10 be	187.94 ± 18.17 ab	257.89 ± 5.10 cf	158.45 ± 27.94 abe	205.41 ± 8.41 ad	30 A	Whisky, nail polish	>1
2-Methyl-1-butanol	85.02 ± 0.82 ab	113.79 ± 5.89 c	75.57 ± 2.64 ad	37.52 ± 19.97 e	100.23 ± 8.49 bc	43.71 ± 8.32 ef	78.32 ± 15.19 ad	111.95 ± 16.87 c	nd	88.36 ± 5.96 ab	61.81 ± 12.48 dg	56.11 ± 5.38 fg	85.81 ± 1.12 ab	43.97 ± 8.03 ef	65.57 ± 2.76 dg	65 A	Pungent odor	>1
2-Ethyl-1-hexanol	0.18 ± 0.03 ab	0.07 ± 0.02 a	0.61 ± 0.05 c	0.37 ± 0.19 bd	nd	1.15 ± 0.39 e	0.40 ± 0.07 bcd	0.06 ± 0.01 a	0.05 ± 0.01 a	0.43 ± 0.05 cd	0.19 ± 0.02 ab	1.21 ± 0.18 e	1.13 ± 0.07 e	0.34 ± 0.03 bd	0.33 ± 0.01 bd	8 A	Mushroom; sweet fruity	0.1–1
3-Methyl-1-pentanol	0.33 ± 0.00 ab	0.37 ± 0.02 ab	0.52 ± 0.07 bc	0.28 ± 0.14 ab	0.52 ± 0.03 bc	0.32 ± 0.11 ab	0.24 ± 0.02 a	2.76 ± 0.37 d	0.35 ± 0.18 ab	0.68 ± 0.04 ce	0.53 ± 0.09 bc	1.44 ± 0.20 f	2.65 ± 0.01 d	0.89 ± 0.12 e	1.83 ± 0.12 g	0.5 A	Alcohol, harsh	>1
4-Methyl-1-pentanol	0.66 ± 0.28 ab	0.28 ± 0.03 cde	0.69 ± 0.16 ab	0.29 ± 0.24 cde	0.14 ± 0.01 d	0.50 ± 0.06 ace	0.32 ± 0.05 cde	0.94 ± 0.28 bf	0.13 ± 0.07 d	nd	0.22 ± 0.05 de	0.56 ± 0.00 ac	0.79 ± 0.24 ab	0.49 ± 0.21 ace	1.07 ± 0.03 f	50 A	Almond, toasted	<0.1
2,3-Butanediol	4.38 ± 0.19 a	2.37 ± 0.29 b	5.09 ± 0.33 a	2.70 ± 1.50 b	2.55 ± 0.47 b	2.71 ± 0.33 b	1.93 ± 0.19 bc	2.76 ± 0.17 b	0.82 ± 0.31 c	4.99 ± 0.01 a	2.68 ± 0.28 b	7.97 ± 1.48 d	12.09 ± 0.70 e	8.69 ± 0.46 d	13.96 ± 1.74 f	150 B	Buttery, creamy	<0.1
Fatty acids	13.05 ± 0.48 ab	5.98 ± 0.03 c	9.96 ± 1.18 de	6.68 ± 3.55 cg	2.34 ± 0.39 f	10.55 ± 2.16 ade	2.27 ± 0.66 f	4.84 ± 0.47 cf	11.80 ± 2.93 abe	8.70 ± 1.22 dg	5.20 ± 0.25 c	11.25 ± 0.74 abde	6.06 ± 0.76 c	5.46 ± 0.76 c	13.27 ± 0.94 b
Butanoic acid	0.03 ± 0.01 ab	0.08 ± 0.01 abcd	0.32 ± 0.00 e	0.20 ± 0.09 f	nd	0.13 ± 0.08 cdf	0.01 ± 0.00 a	0.11 ± 0.01 bcd	0.06 ± 0.03 abcd	0.05 ± 0.02 abc	nd	0.02 ± 0.00 a	0.14 ± 0.11 df	nd	0.01 ± 0.00 a	10 A	Cheese	<1
Decanoic acid	0.90 ± 0.05 a	0.52 ± 0.01 bcd	0.34 ± 0.06 bce	0.33 ± 0.16 bce	0.06 ± 0.01 e	0.59 ± 0.20 bcd	0.14 ± 0.03 e	0.24 ± 0.01 be	2.40 ± 0.66 f	0.79 ± 0.07 ad	0.34 ± 0.02 bce	0.65 ± 0.07 acd	0.26 ± 0.06 be	0.29 ± 0.05 bce	0.86 ± 0.05 ad	15 A	Fatty, unpleasant	<0.1
Hexanoic acid	4.40 ± 0.12 a	1.51 ± 0.03 bcd	3.44 ± 0.41 fg	2.46 ± 1.34 eh	1.11 ± 0.19 bd	3.54 ± 0.57 fg	0.79 ± 0.16 d	1.69 ± 0.18 bch	1.22 ± 0.83 bd	2.84 ± 0.16 ef	2.01 ± 0.02 bceh	3.53 ± 0.33 fg	2.24 ± 0.24 ceh	1.70 ± 0.24 bch	4.19 ± 0.38 ag	3 B	Cheese, fatty	>1
Octanoic acid	7.22 ± 0.37 ab	3.51 ± 0.04 cd	5.40 ± 0.75 ef	3.53 ± 1.86 cd	1.16 ± 0.19 g	6.07 ± 1.44 af	1.25 ± 0.49 gh	2.69 ± 0.29 ch	8.12 ± 1.41 b	4.26 ± 0.95 de	2.65 ± 0.30 ch	6.78 ± 0.31 abf	2.73 ± 0.57 ceh	3.35 ± 0.43 cd	7.83 ± 0.50 b	0.5 A	Cheese, rancid, harsh	>1
Isobutyric acid	0.27 ± 0.02 a	0.19 ± 0.05 b	0.22 ± 0.02 bc	0.09 ± 0.06 de	nd	0.11 ± 0.03 d	0.08 ± 0.02 de	0.11 ± 0.00 d	nd	0.39 ± 0.00 f	0.08 ± 0.02 de	0.10 ± 0.01 de	0.24 ± 0.03 ac	0.06 ± 0.00 e	0.11 ± 0.02 d	2.3 B	Cheese, butter, rancid	<0.1
Isovaleric acid	0.23 ± 0.03 a	0.18 ± 0.02 b	0.25 ± 0.02 a	0.07 ± 0.04 cd	nd	0.12 ± 0.01 c	nd	nd	nd	0.37 ± 0.01 e	0.11 ± 0.03 c	0.17 ± 0.02 b	0.45 ± 0.02 f	0.06 ± 0.04 d	0.27 ± 0.02 a	0.0334 A	Fatty, sweet	>1
Acetates	240.75 ± 8.61 ab	202.96 ± 12.81 ab	92.31 ± 37.42 cd	84.45 ± 48.89 cd	92.83 ± 23.27 cd	45.27 ± 23.16 c	57.73 ± 17.26 c	225.88 ± 123.01 ab	94.18 ± 26.13 cd	101.88 ± 42.84 cd	58.18 ± 18.82 c	169.14 ± 12.17 ad	81.42 ± 5.60 cd	197.03 ± 44.92 ab	279.77 ± 133 b
Ethyl acetate	59.68 ± 2.36 a	57.27 ± 4.62 a	39.08 ± 5.48 bc	29.97 ± 16.75 bcd	67.16 ± 14.30 ae	19.14 ± 4.78 d	21.34 ± 1.95 d	61.72 ± 14.61 ae	18.34 ± 1.68 d	29.55 ± 4.05 bcd	27.26 ± 1.36 bd	43.19 ± 2.03 c	27.51 ± 0.46 bd	40.26 ± 9.17 bc	74.62 ± 8.26 e	7.5 A	Fruity	>1
Butyl acetate	1.48 ± 0.05 abc	0.20 ± 0.02 a	3.35 ± 1.57 cd	2.64 ± 1.52 bc	nd	4.97 ± 2.93 d	2.34 ± 0.82 abc	0.41 ± 0.20 a	nd	2.79 ± 1.27 bc	1.09 ± 0.43 ab	7.15 ± 0.69 e	7.64 ± 0.53 e	2.93 ± 0.69 bcd	1.63 ± 0.80 abc	1.6 B	Pleasant, fruity	>1
Hexyl acetate	1.88 ± 0.00 a	0.78 ± 0.01 bc	0.55 ± 0.33 bc	0.41 ± 0.24 bc	0.10 ± 0.03 c	0.33 ± 0.24 bc	0.32 ± 0.12 bc	0.97 ± 0.62 bd	0.54 ± 0.17 bc	0.63 ± 0.30 bc	0.20 ± 0.08 c	1.04 ± 0.05 bd	0.52 ± 0.07 bc	1.51 ± 0.33 ad	1.74 ± 1.16 a	1.5 A	Pleasant, fruity, pear	0.1–1
Isobutyl acetate	1.32 ± 0.03 a	1.15 ± 0.06 ab	0.35 ± 0.15 c	0.32 ± 0.18 c	0.21 ± 0.07 c	0.17 ± 0.09 c	0.27 ± 0.08 c	0.84 ± 0.46 abc	0.57 ± 0.07 bc	0.65 ± 0.27 abc	0.38 ± 0.13 c	0.60 ± 0.05 bc	0.31 ± 0.00 c	0.78 ± 0.21 abc	1.34 ± 0.61 a	1.6 A	Fruity, apple, banana	0.1–1
Isoamyl acetate	159.30 ± 5.57 a	131.16 ± 7.55 acd	45.17 ± 27.61 be	47.1 ± 27.79 be	23.05 ± 8.10 e	19.08 ± 13.95 e	30.70 ± 16.75 be	148.19 ± 97.84 ad	74.72 ± 24.21 bcde	61.73 ± 33.50 bce	26.62 ± 15.40 e	109.86 ± 8.68 abcd	41.97 ± 4.21 be	142.25 ± 32.35 ad	184.77 ± 112.22 a	0.03 A	Banana, fruity, sweet	>1
2-Methybutyl acetate	17.07 ± 0.61 a	12.41 ± 0.58 abc	3.80 ± 2.29 d	4.02 ± 2.42 d	2.32 ± 0.77 d	1.59 ± 1.16 d	2.76 ± 1.44 d	13.75 ± 9.27 abc	nd	6.52 ± 3.44 bd	2.63 ± 1.41 d	7.30 ± 0.67 bd	3.47 ± 0.33 d	9.29 ± 2.18 bcd	15.68 ± 9.96 ac	0.02~0.05 A	Fruity, comfort fat	>1
.1Ethyl esters	478.95 ± 15.94 a	270.43 ± 5.26 bcd	202.36 ± 63.10 bce	261.46 ± 155.03 bcd	190.05 ± 43.89 bce	210.60 ± 128.38 bce	80.87 ± 32.41 e	209.05 ± 93.65 bce	132.04 ± 32.14 be	175.82 ± 54.74 bce	129.27 ± 19.66 be	289.89 ± 16.02 cd	138.79 ± 11.83 bce	196.39 ± 42.52 bce	314.15 ± 167.43 d
Ethyl caprate	101.47 ± 3.59 a	56.60 ± 0.91 bc	41.10 ± 3.99 bc	60.17 ± 36.04 abc	19.82 ± 1.13 c	48.72 ± 25.73 bc	17.85 ± 6.39 c	37.31 ± 8.33 bc	31.94 ± 5.51 bc	49.81 ± 8.93 bc	34.29 ± 4.67 bc	69.73 ± 3.05 ab	27.96 ± 0.31 bc	36.24 ± 7.60 bc	64.64 ± 27.20 ab	0.2 B	Sweet, fruity	>1
Ethyl butyrate	3.26 ± 0.08 ab	1.90 ± 0.13 acde	2.05 ± 1.09 acde	2.00 ± 1.15 acde	4.56 ± 1.48 acde	0.87 ± 0.53 de	0.50 ± 0.15 d	2.82 ± 1.73 ac	3.07 ± 1.04 ab	1.15 ± 0.50 de	0.93 ± 0.44 de	1.01 ± 0.14 de	0.81 ± 0.05 de	1.36 ± 0.33 cde	2.22 ± 1.13 ace	0.02 A	Strawberry, apple	>1
Ethyl isobutyrate	0.08 ± 0.00 ab	0.11 ± 0.02 b	0.03 ± 0.00 c	0.02 ± 0.01 ac	nd	nd	0.02 ± 0.01 c	0.05 ± 0.03 ac	nd	0.06 ± 0.05 abc	0.02 ± 0.01 ac	nd	nd	0.03 ± 0.02 ac	0.03 ± 0.01 ac	0.015 B	Strawberry	>1
Ethyl dodecanoate	7.36 ± 0.11 a	4.09 ± 0.13 bcd	4.29 ± 0.27 bcd	5.88 ± 3.10 ab	0.93 ± 0.10 f	4.32 ± 1.78 bcd	1.78 ± 0.54 ef	2.90 ± 0.31 de	4.07 ± 0.02 bcd	5.80 ± 0.47 ab	3.32 ± 0.50 cde	4.87 ± 0.18 bc	2.63 ± 0.05 def	2.42 ± 0.42 def	5.16 ± 1.49 bc	0.5 A	Sweet, floral, fruity	>1
Ethyl propionate	0.89 ± 0.01 a	0.72 ± 0.08 ab	0.29 ± 0.11 cd	0.19 ± 0.10 cd	0.24 ± 0.08 cd	0.08 ± 0.04 c	0.18 ± 0.01 cd	0.44 ± 0.20 bde	0.08 ± 0.01 c	0.60 ± 0.20 abe	0.41 ± 0.12 de	0.19 ± 0.05 cd	0.22 ± 0.01 cd	0.21 ± 0.04 cd	0.45 ± 0.13 bde	1.8 B	Banana; apple	0.1–1
Ethyl nonanoate	1.12 ± 0.02 a	0.54 ± 0.03 bcd	0.27 ± 0.04 bd	0.37 ± 0.22 bd	0.17 ± 0.01 d	0.31 ± 0.19 bd	0.14 ± 0.04 d	0.34 ± 0.12 bd	0.45 ± 0.07 bd	0.91 ± 0.22 ac	0.37 ± 0.04 bd	0.59 ± 0.00 bc	0.41 ± 0.04 bd	0.38 ± 0.07 bd	0.62 ± 0.32 bc	1.3 B	Banana, grape	0.1–1
Ethyl hexanoate	92.45 ± 2.35 a	44.8 ± 1.14 bc	30.01 ± 17.87 bcd	43.10 ± 25.48 bcd	44.1 ± 15.53 bc	22.96 ± 17.09 bcd	10.19 ± 4.90 d	44.34 ± 31.14 bc	32.87 ± 9.57 bcd	24.16 ± 13.31 bcd	16.53 ± 9.68 bd	33.84 ± 2.34 bcd	21.29 ± 2.52 bd	37.16 ± 8.10 bcd	55.35 ± 39.22 c	0.003 B	Banana, sweet fruity	>1
Ethyl heptanoate	0.38 ± 0.01 a	0.12 ± 0.01 bcd	0.10 ± 0.05 bcd	0.10 ± 0.06 bcd	0.06 ± 0.04 d	0.07 ± 0.05 bd	0.05 ± 0.01 d	0.08 ± 0.04 bd	0.10 ± 0.03 bcd	0.17 ± 0.05 c	0.07 ± 0.04 bd	0.10 ± 0.00 bcd	0.09 ± 0.01 bd	0.07 ± 0.02 bd	0.15 ± 0.09 bc	0.22 B	Pineapple, fruity	0.1–1
Ethyl tetradecanoate	0.13 ± 0.00 ab	0.09 ± 0.00 cde	0.08 ± 0.00 ce	0.09 ± 0.04 cde	0.02 ± 0.00 d	0.06 ± 0.02 cde	0.06 ± 0.01 ce	0.06 ± 0.00 cde	0.26 ± 0.01 f	0.16 ± 0.01 b	0.13 ± 0.02 ab	0.09 ± 0.01 cde	0.06 ± 0.02 cde	0.05 ± 0.01 de	0.10 ± 0.00 ac	2 B	Mild waxy, soapy	0.1–1
Ethyl hexadecanoate	0.17 ± 0.01 a	0.17 ± 0.00 a	0.11 ± 0.01 ab	0.10 ± 0.06 ab	nd	0.09 ± 0.06 ab	0.07 ± 0.01 b	0.09 ± 0.03 ab	0.12 ± 0.01 ab	0.16 ± 0.01 ab	0.15 ± 0.00 ab	0.15 ± 0.01 ab	0.07 ± 0.02 b	0.12 ± 0.05 ab	0.13 ± 0.05 ab	1.5 B	Fruity, sweet, fatty	0.1–1
Ethyl octanoate	268.88 ± 9.71 a	159.45 ± 4.92 bcd	122.20 ± 39.52 bcde	147.82 ± 87.82 bcd	120.13 ± 27.99 bcde	132.17 ± 82.41 bcde	48.48 ± 19.73 e	120.60 ± 51.72 bcd	57.00 ± 15.76 e	88.08 ± 30.22 bce	72.13 ± 14.77 be	176.44 ± 10.20 cd	84.42 ± 8.80 be	117.72 ± 25.77 bcde	182.21 ± 96.53 d	0.002 B	Floral, pineapple, pear	>1
Ethyl 3-hydroxybutanoate	0.05 ± 0.00 ab	0.09 ± 0.06 bc	0.03 ± 0.00 ab	0.03 ± 0.01 ab	0.02 ± 0.01 ab	0.06 ± 0.01 ab	nd	0.03 ± 0.00 ab	0.05 ± 0.01 ab	nd	0.02 ± 0.00 ab	0.13 ± 0.10 a	0.03 ± 0.01 c	0.01 ± 0.01 ab	0.05 ± 0.04 a	20 B	Sweet fruity, grape	<0.1
Ethyl 9-decenoate	2.71 ± 0.08 a	1.75 ± 0.06 ab	1.81 ± 0.14 ab	1.59 ± 0.95 ab	nd	0.89 ± 0.46 a	1.54 ± 0.62 ab	nd	2.03 ± 0.14 ab	4.76 ± 0.79 c	0.90 ± 0.14 b	2.75 ± 0.15 a	0.81 ± 0.01 b	0.61 ± 0.11 b	3.06 ± 1.23 a	0.1 B	Fruity	>1
Other esters	2.62 ± 0.08 a	1.54 ± 0.01 abcd	1.15 ± 0.14 bcde	1.44 ± 0.83 bcde	0.60 ± 0.06 be	1.18 ± 0.56 bcde	0.44 ± 0.14 e	1.26 ± 0.35 bcde	0.88 ± 0.13 bcde	1.23 ± 0.27 bcde	0.71 ± 0.00 bce	1.76 ± 0.18 acd	0.84 ± 0.10 bcde	0.78 ± 0.19 bce	1.93 ± 0.83 ad
Diethyl succinate	0.40 ± 0.01 a	0.17 ± 0.02 bc	0.17 ± 0.01 bcd	0.14 ± 0.07 bc	0.11 ± 0.03 bc	0.19 ± 0.01 bd	0.07 ± 0.02 bc	0.05 ± 0.03 d	0.08 ± 0.01 bc	0.11 ± 0.02 bc	0.05 ± 0.00 d	0.35 ± 0.11 a	0.13 ± 0.04 bc	0.08 ± 0.02 bc	0.30 ± 0.06 ad	200 B	Light fruity, wine	<0.1
Isopentyl hexanoate	0.91 ± 0.04 a	0.54 ± 0.00 abc	0.36 ± 0.11 bc	0.46 ± 0.27 abc	0.27 ± 0.05 bc	0.31 ± 0.23 bc	0.13 ± 0.05 c	0.50 ± 0.23 abc	0.29 ± 0.06 bc	0.37 ± 0.16 bc	0.20 ± 0.04 bc	0.49 ± 0.02 abc	0.26 ± 0.04 bc	0.27 ± 0.10 bc	0.63 ± 0.35 ab	0.32 D	Fennel, fruity, spices	>1
3-Methylbutyl octanoate	1.31 ± 0.03 a	0.83 ± 0.04 abc	0.62 ± 0.03 bc	0.84 ± 0.00 abc	0.22 ± 0.02 c	0.68 ± 0.32 bc	0.24 ± 0.07 c	0.71 ± 0.14 bc	0.51 ± 0.07 bc	0.75 ± 0.13 abc	0.46 ± 0.04 bc	0.93 ± 0.05 abc	0.45 ± 0.02 bc	0.43 ± 0.07 bc	1.00 ± 0.41 abc	0.125 D	Sweet	>1
Benzene derivatives	137.28 ± 8.94 a	138.93 ± 12.40 a	71.02 ± 1.59 cd	37.35 ± 20.79 bg	20.52 ± 5.48 g	60.61 ± 15.26 cd	53.86 ± 13.02 bc	88.82 ± 6.24 ef	40.72 ± 0.40 b	74.93 ± 0.36 de	89.38 ± 4.89 ef	148.21 ± 6.48 a	91.14 ± 11.28 ef	95.62 ± 13.99 f	134.75 ± 2.12 a
Benzyl alcohol	0.12 ± 0.05 ab	0.08 ± 0.01 acd	0.07 ± 0.01 acd	0.05 ± 0.04 cd	nd	0.05 ± 0.00 cd	0.05 ± 0.00 cd	0.04 ± 0.00 cd	0.05 ± 0.00 cd	0.08 ± 0.06 cd	0.03 ± 0.01 acd	0.08 ± 0.01 c	0.07 ± 0.03 abd	0.07 ± 0.00 cd	0.12 ± 0.03 cd	200 A	Almond	<0.1
Benzaldehyde	0.12 ± 0.01 ab	0.09 ± 0.00 a	0.20 ± 0.05 c	nd	nd	0.17 ± 0.01 bc	0.10 ± 0.01 a	0.26 ± 0.03 de	0.11 ± 0.04 a	0.21 ± 0.02 c	0.09 ± 0.01 a	0.22 ± 0.04 cd	0.21 ± 0.02 c	0.19 ± 0.04 c	0.28 ± 0.03 e	0.3 B	Sweet, ferity	0.1–1
Benzene acetaldehyde	0.05 ± 0.01 a	0.07 ± 0.03 ab	0.04 ± 0.03 a	0.05 ± 0.03 a	nd	0.16 ± 0.13 c	nd	0.05 ± 0.00 ab	0.02 ± 0.00 a	0.07 ± 0.02 ab	0.05 ± 0.01 a	0.06 ± 0.01 ab	0.07 ± 0.00 ab	0.06 ± 0.01 ab	0.13 ± 0.04 bc	0.004 A	Sugar, floral	>1
Phenylethyl alcohol	108.40 ± 7.57 ab	117.85 ± 10.97 b	55.46 ± 2.00 cd	31.53 ± 17.85 fg	19.90 ± 5.40 f	45.38 ± 10.74 cg	37.13 ± 8.11 g	67.78 ± 4.17 de	32.60 ± 1.14 fg	nd	77.17 ± 5.16 e	99.59 ± 3.62 a	77.03 ± 10.27 e	67.74 ± 9.12 de	98.23 ± 0.05 a	10 A	Rose, pollen, perfume	>1
2-Phenethyl acetate	28.60 ± 1.33 a	20.84 ± 1.40 b	15.25 ± 0.37 c	5.72 ± 2.87 d	0.63 ± 0.08 f	14.85 ± 4.63 c	16.58 ± 4.91 be	20.69 ± 2.03 b	7.93 ± 0.71 de	74.58 ± 0.34 i	12.04 ± 0.27 ce	48.26 ± 2.82 h	13.77 ± 0.95 c	27.58 ± 4.85 a	35.99 ± 2.20 f	0.25 A	Floral, pleasant	>1
Aldoketones	1.81 ± 0.19 aef	1.14 ± 0.17 bc	1.52 ± 0.28 ace	0.96 ± 0.45 bc	0.04 ± 0.00 d	1.22 ± 0.06 bc	0.78 ± 0.04 b	1.28 ± 0.16 abc	0.09 ± 0.01 d	2.20 ± 0.13 f	0.82 ± 0.02 b	4.21 ± 0.83 g	1.85 ± 0.12 ef	1.96 ± 0.59 ef	4.33 ± 0.08 g
Decanal	0.11 ± 0.02 abc	0.07 ± 0.00 abc	0.17 ± 0.06 bcd	0.05 ± 0.04 ab	0.04 ± 0.00 a	0.12 ± 0.08 abc	0.03 ± 0.00 a	0.07 ± 0.02 abc	0.09 ± 0.01 abc	0.07 ± 0.00 abc	0.03 ± 0.01 a	0.17 ± 0.03 cd	0.23 ± 0.02 d	0.05 ± 0.01 ab	0.14 ± 0.09 abcd	0.00125 C	Soap, orange peel	>1
Nonanal	0.04 ± 0.01 a	0.05 ± 0.01 ab	0.08 ± 0.00 bc	0.10 ± 0.01 c	nd	0.08 ± 0.06 bc	0.05 ± 0.01 ab	0.05 ± 0.01 ab	nd	nd	0.02 ± 0.00 a	0.17 ± 0.02 d	0.16 ± 0.01 d	0.02 ± 0.01 a	0.02 ± 0.00 a	0.015 C	Green, slightly pungent	>1
2,3-Butanedione	1.67 ± 0.22 abc	1.02 ± 0.15 cd	1.27 ± 0.22 cde	0.81 ± 0.40 c	nd	1.03 ± 0.08 cd	0.70 ± 0.05 c	1.16 ± 0.13 cde	nd	2.13 ± 0.12 c	0.77 ± 0.02 c	3.88 ± 0.79 f	1.46 ± 0.09 abd	1.89 ± 0.58 bc	4.16 ± 0.00 f	0.2~0.3 D	Cream, essences	>1
Total aroma	1149.74 ± 36. 19 d	996.60 ± 40.03 cd	665.90 ± 113.42 ab	551.25 ± 313.65 a	731.32 ± 88.85 abc	502.78 ± 202.62 a	485.21 ± 118.64 a	956.22 ± 285.42 bcd	495.02 ± 91.52 a	659.95 ± 116.38 ab	503.87 ± 87.49 a	889.34 ± 62.99 bcd	692.28 ± 35.87 ab	717.05 ± 140.88 abc	1029.04 ± 318.19 d

Note: Values with different lowercase letters in the same row are significantly different according to the Duncan test (p < 0.05). nd indicates not detected, NF indicates not found, *: mg/L. A: [41]; B: [42]; C: [43] D: [44].

, the 485.23~1149.74 mg/L volatile compounds were detected from young wines, which included 10 varietal aroma compounds and 47 fermentative aroma compounds. The former contained C₆ and terpene compounds, and the later contained higher alcohols, fatty acids, esters, benzene derivatives, and aldoketones.

Varietal Aromas Compounds of Wines

The C₆ compound contents in SCw (0.42~1.10 mg/L) were slightly lower than those in FSCw (1.33 mg/L) (Table 2 and Figure 5A), which mainly presented an unpleasant green odor in the wines. The terpene contents in the wines were 0.01~0.66 mg/L, and F4-13w (0.66 mg/L) and F5-18w (0.64 mg/L) generated higher terpene contents than FSCw (0.56 mg/L) (Figure 5B). Terpenes with floral and fruity traits had a positive effect on the varietal aroma of the wines [41,45]. Linalool, cis-geraniol, and geranyl acetone were the odor-active compounds (OAV ≥ 1) of the wines (Table 2). Liu, et al. [46] reported that the selected indigenous S. cerevisiae strains improved terpene synthesis and contributed a pleasant aroma to the wines. These results mean that the selected S. cerevisiae strains slightly decreased the green flavor of wine, and S. cerevisiae F4-13 and F5-18 had great potential in highlighting the varietal aroma characteristics of wines.

Fermentative Aroma Compounds of Wines

As shown in Table 2, FSCw had the highest content of fermentative aroma compounds (1147.85 mg/L), followed by MXY-19w (1044.55 mg/L), F4-13w (994.90 mg/L) and F5-21w (955.18 mg/L), while the other SCw had 42% to 77% of that in FSCw.

Proper higher alcohol content contributes to mellow, fruity, and floral traits in wines, but excessive higher alcohols (>400 mg/L) causes pungent odors and unpleasant smells [47]. Moreover, higher alcohols are also the precursors of ester biosynthesis, which contributes the fruity trait [48]. The higher alcohol contents in most SCw (157.88~373.92 mg/L) were lower than 400 mg/L except for F5-12w (424.30 mg/L) and F5-21w (424.05 mg/L), and those in F4-13w, F5-7w, F5-19w, F6-23w, HT-10w, and MXY-19w (286.57~373.92 mg/L) were higher than that in FSCw (273.39 mg/L) (Figure 5C). As shown in Table 2, the isoamylol, 2-methyl-1-butanol and 3-methyl-1-pentanol were the odor active higher alcohols (OAV > 1), which contributed to the organoleptic properties of the wines.

Volatile fatty acids mainly present wax, butter, and sweat traits [47]. The volatile fatty acid content in SCw (2.2~11.80 mg/L) except for MXY-19w (13.27 mg/L) was lower than that in FSCw (13.05 mg/L) (Figure 5D), and octanoic acid and hexanoic acid were the odor-active fatty acids (OAV > 1) (Table 2).

Esters mainly contribute fruity and floral traits to wines, and include acetates, ethyl esters, and other esters [9]. Compared with that in FSCw (240.75 mg/L), the acetate content in MXY-19w (279.77 mg/L) was increased, that in F5-21w (225.88 mg/L), F4-13w (202.96 mg/L) and ZXY-17w (197.03 mg/L) showed an insignificant difference, and those in other SCw (45.27~169.14 mg/L) were decreased. Ethyl acetate, isoamyl acetate, and 2-methylbutyl acetate were the main odor-active acetates (OAV > 1) (Figure 5E). The ethyl ester contents in SCw (80.87~314.15 mg/L) were significantly lower than in FSCw (478.95 mg/L) (Figure 5F). Ethyl octanoate, ethyl hexanoate, and ethyl caprate were the main odor active ethyl esters (OAV > 1). The other ester contents in F4-13w (1.54 mg/L), SXY-4w (1.76 mg/L), and MXY-19w (1.93 mg/L) were insignificantly different from those in FSCw (2.62 mg/L) (Figure 5G), but those in other SCw (0.44~1.44 mg/L) were lower than those in FSCw. Isopentyl hexanoate and 3-methylbutyl octanoate were the main odor esters (OAV > 1).

Benzene derivative mainly contribute rose, strawberry, and pleasant traits to wine [49]. Benzene derivative contents in F4-13w (138.93 mg/L), SXY-4w (148.21 mg/L) and MXY-19w (134.75 mg/L) were insignificantly different from that in FSCw (137.28 mg/L), while those in other SCw (20.52~95.62 mg/L) were lower than those in FSCw (Figure 5H). Phenylethyl alcohol, 2-phenethyl acetate, and benzene acetaldehyde were the main odor-active benzene derivatives (OVA > 1).

Aldoketones, including aldehydes and ketones, mainly contribute nut, fat, and butter traits to wine [50]. The aldoketone contents in MXY-19w, SXY-4w, F6-23w, ZXY-17w, and HT-10w (1.85~4.33 mg/L) were higher than those in FSCw (1.81 mg/L), while those in other SCw (0.04~1.52 mg/L) were lower than in FSCw (Figure 5I).

As charted in Figure 6, hierarchical clustering analysis of the volatile compound content in SCw indicated that all the SCw were classified into three categories. MXY-19w, SXY-4w, FSCw, and F4-13w were clustered together with the highest contents of aroma compounds. F6-23w, HT-10w, F5-18w, F5-7w, and F5-9w were clustered together with medium contents of aroma compounds. F6-8w, F5-12w, F5-21w, ZXY-17w, F9-23w, and F5-19w were grouped together with the lowest contents of aroma compounds.

3.3.4. Sensory Evaluation of Wines

The sensory evaluation of SCw indicated that red fruity, black fruity, floral, green, spices, and yeasty were the main aroma traits of wines (Figure 7). Compared with those in FSCw, the red fruity trait in F4-13w, F5-21w, F5-18w, F5-12w, HT-10w, MXY-19w, F6-8w, F6-23w, and ZXY-17w was improved, especially in F4-13w, the floral trait was enhanced in F4-13w and SXY-4w, and the green trait with negative odor in SCw was reduced [51]. The spices trait in SCw was almost the same as that in FSCw, apart from F9-23w, F5-12w, F5-19w, and MXY-19w. However, MXY-19w, F9-23w, and SXY-4w presented a mild yeasty trait, which exhibited strong flocculation ability (Figure 3D). Bauer, et al. [52] indicated that strong flocculation ability led to the early deposition of yeast cells during alcoholic fermentation, which resulted in yeasty traits in wines.

4. Conclusions

A total of 199 S. cerevisiae strains were isolated and identified from the vineyards and spontaneous fermentation must at eastern foot of Helan Mountain in China, and spontaneous fermentation must was the best source for collecting S. cerevisiae. Among the S. cerevisiae strains, 14 isolates exhibited excellent tolerance abilities to 250 mg/L sugar, 100 mg/L SO₂, and 12% (v/v) ethanol. F4-13 had the better oenological properties, with low H₂S production (+), suitable flocculation ability (58.74%), and a certain L-malic-acid-reducing ability (49.07%), and generated high contents of polyphenol, anthocyanin, tannin, terpenes, and higher alcohols in wines, which resulted in the improvement of the red fruity and floral aromas of the wines, suggesting that F4-13 had the potential to be a starter for winemaking. The presented results provide a strategy for the selection of indigenous S. cerevisiae for wine fermentation to produce high-quality wine with regional characteristics. However, a large-scale wine fermentation characteristic of F4-13 should be carried out in the future.