Food Bioscience 55 (2023) 102952

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Sequential fermentation with indigenous non-Saccharomyces yeasts and

Saccharomyces cerevisiae for flavor and quality enhancement of Longyan dry
white wine
Xiaodi Wang a, Jiawei Chen b, Xiaoxin Ge a, Xiaofang Fu b, Chao Dang a, c, Jie Wang a, c,
Yaqiong Liu a, c, *
a
College of Food Science and Technology, Hebei Agricultural University, Baoding, 071000, China
b
China Great Wall Wine Co., Ltd., Zhangjiakou, 075400, China
c
Hebei Technology Innovation Center of Agricultural Products Processing, Baoding, 071000, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, we investigated the effect of the sequential fermentation of four selected indigenous non-Saccha­
Longyan wine romyces yeasts (Candida stellimalicola HL-S-5, Rhodotorula mucilaginosa HL-S-8, Hanseniaspora uvarum HL-S-9, and
Sequential fermentation Pichia kluyveri XL-48-6) and Saccharomyces cerevisiae VL2 on the oenological parameters, volatile compositions,
Native non-saccharomyces yeasts
organic acids, and sensory characteristics of Longyan dry white wine. Results showed that the ethanol concen­
Volatile compounds
tration of pure fermentation (11.12 ± 0.04%, v/v) was the highest among all fermentations. Furthermore, the
Organic acids
aroma concentrations produced by the fermentation of R. mucilaginosa HL-S-8/VL2 (9929.35 ± 96.87 μg/L) and
P. kluyveri XL-48-6/VL2 (9017.67 ± 85.91 μg/L) were significantly higher than that of VL2 (7654.64 ± 397.07
μg/L). In particular, the treatment of R. mucilaginosa HL-S-8/VL2 increased the content of acetic esters (512.11 ±
12.92 μg/L) and fatty acid ethyl esters (6893.64 ± 56.67 μg/L), which enhanced fruity and floral characteristics.
The sequential fermentation of R. mucilaginosa HL-S-8/VL2 led to significantly higher organic acid content
(3843.32 ± 73.93 mg/L) than the other samples. Moreover, the sequential fermentation of R. mucilaginosa HL-S-
8/VL2 wine samples resulted in the highest scores in sensory evaluation. Therefore, the sequential fermentation
of R. mucilaginosa HL-S-8/VL2 was an effective way to improve wine flavor and quality. This work will provide a
useful reference for improving the flavor and quality of Longyan wine through non-Saccharomyces cerevisiae.

1. Introduction
Longyan grape is an ancient characteristic white grape variety in
China, and it has been cultivated for over 800 years (Wang et al., 2022).
This grape has thin and transparent skin and a juicy texture; it is
exceedingly tasty, so it is not only superior for fresh consumption but
also as a high-quality raw material for winemaking. The Huailai region
vineyard (40◦ N, 115◦ E) in Hebei Province is the most suitable for the
growth of Longyan grape. This region is a unique “V”-type basin be­
tween two mountains and one reservoir with slightly acidic soil; it fea­
tures a large temperature difference between day and night, sufficient
light, and a hot rainy season (Sun et al., 2018). The first dry white wine
in China was made from Longyan grape, which was highly popular
among consumers for its mellow taste and pleasant fruit flavor.
Commercial yeasts are widely used in large-scale wine production
due to their ease of use and stable fermentation characteristics (Ge et al.,
2022), but the complexity and typicality of their flavor are poor
compared with successful natural fermentation (Shi et al., 2019). This is
due to the existence of a variety of indigenous non-Saccharomyces yeasts
in must during natural fermentation, which can contribute to wine
aroma and stylistic distinction, but may cause deterioration of the wine
(Liu et al., 2016). However, the difficulty with which non-Saccharomyces
yeast finishes alcoholic fermentation requires combined fermentation
with Saccharomyces cerevisiae. Therefore, the co-fermentation of non-­
Saccharomyces yeast and S. cerevisiae to improve wine flavor and quality
has received increasing recognition (Padilla et al., 2016).
In previous studies, non-Saccharomyces yeast strains with different
oenological potentials were used to modulate the aroma and organic
acid profile of white wine. For example, the co-fermentation of P.
kluyveri and S. cerevisiae was found to improve the varietal thiol

* Corresponding author. College of Food Science and Technology, Hebei Agricultural University, Baoding, 071000, China.
E-mail address: shplyq@hebau.edu.cn (Y. Liu).

https://doi.org/10.1016/j.fbio.2023.102952
Received 4 May 2023; Received in revised form 27 June 2023; Accepted 16 July 2023
Available online 19 July 2023
2212-4292/© 2023 Elsevier Ltd. All rights reserved.
X. Wang et al.
concentrations compared with their pure fermentation, leading to Sau­
vignon Blanc’s distinctive Marlborough style (Anfang et al., 2009). The
sequential fermentation of C. stellimalicola and S. cerevisiae led to the
highest total concentration of esters, higher alcohol, and terpenols in
Muscat d’ Alexandrie wine (Rodriguez et al., 2010). Hu et al. (2018)
reported that sequential fermentation of H. uvarum and S. cerevisiae can
improve the fruity and floral profiles of Ecolly wine. Moreover, the lactic
acid content of Petit Manseng wine fermented sequentially with non-­
Saccharomyces yeast and S. cerevisiae increased by 490 μg/L compared
with that under pure fermentation (Wang et al., 2022). The sequential
fermentation of Torulaspora delbrueckii and S. cerevisiae has low malic
acid and high lactic acid contents than pure fermentation in Chardonnay
wine (Puertas et al., 2018). Indigenous non-Saccharomyces yeasts with
excellent oenological characteristics have high environment adapt­
ability, and considered as key factors for the ‘terroir’ characteristics of
regional wine (Zhang et al., 2021). However, studies about the contri­
butions of these indigenous non-Saccharomyces yeast strains in
improving wine aroma and quality for Longyan dry white wine are
limited.
This study aimed to investigate the effect of sequential fermentation
of four data-unpublished indigenous non-Saccharomyces yeasts
(C. stellimalicola HL-S-5, R. mucilaginosa HL-S-8, H. uvarum HL-S-9, and
P. kluyveri XL-48-6) and S. cerevisiae VL2 on the oenological parameters,
volatile compositions, organic acids, and sensory characteristics of
Longyan dry white wine. Our results not only highlight the role of non-
Saccharomyces yeasts in improving wine flavor and quality but also
provide a reference for the selection of yeast for fermentation produc­
tion of Longyan dry white wine.
2. Materials and methods
2.1. Yeast and winemaking
Longyan grapes were harvested from the Huailai region vineyard
(40◦ 17′57′′N, 115◦ 25′56′′E, Hebei Province, China) on October 10, 2021,
and fermentation experiments were carried out in China Great Wall
Wine Co., Ltd. Grape juice (162.34 g/L residual sugar and 8.6 g/L total
acidity expressed as tartaric acid) was obtained after squeezing by a
nitrogen-protected airbag, during which pectinase (15 g/t, LAFAZYM®
EXTRACT) and potassium metabisulfite (80 g/t) were added into the
grape juice. The grape juice was stored at 6 ◦ C–7 ◦ C for 48 h to separate
the clarified must and collected for subsequent fermentation experi­
ments. Distributed 60 L grape must into fifteen 5-L volume cylinder glass
container with a working volume of 4 L. There were five groups with
three replicates in each group for inoculated fermentation. Control
group was the pure fermentation of S. cerevisiae VL2 (200 g/t, ZYMA­
FLORE® VL2 from LAFFORT, France), and the four other groups were
the sequential fermentations of C. stellimalicola HL-S-5/VL2,
R. mucilaginosa HL-S-8/VL2, H. uvarum HL-S-9/VL2, and P. kluyveri
XL-48-6/VL2, respectively. These four indigenous non-Saccharomyces
yeasts were screened from the Huailai region Longyan vineyards and
preserved in college of Food Science and Technology of Hebei Agricul­
tural University. In sequential fermentation, non-Saccharomyces yeast
was inoculated at 106 CFU/mL to ferment for 24 h before the inoculation
of S. cerevisiae. Fermentation was considered complete when the resid­
ual sugar level was less than 4 g/L. Samples (200 mL) were collected
from the early stage (residual sugar remaining 80%), middle stage (re­
sidual sugar remaining 50%), late stage (residual sugar remaining 15%),
and end stage (residual sugar less than 4 g/L) and stored at − 20 ◦ C until
analysis. All fermentations in this study were performed in triplicate.
2.2. Oenological parameters analysis
The wine oenological parameters were monitored by Fourier
Transform Infrared Spectrophotometer (Wine Scan FT120, FOSS A/S,
Hilleroed, Denmark) through a 32 μm path length cuvette in the China
2
Food Bioscience 55 (2023) 102952
Great Wall Wine Co., Ltd. The company provides ready-made wine to
calibrate the apparatus. After samples were centrifuged at 6201×g
(8000 rpm, TGL21M, Hunan Yida Jinghua Instrument Co., Ltd, Hunan,
China) at 4 ◦ C for 15 min by scanning the supernatant on the instrument
from wave numbers 926 cm− 1 to 5012 cm− 1 at 4 cm− 1 intervals, the
basic oenological parameters of all samples were obtained, including
total sugar (g/L), total acids (g/L), and ethanol content (%, v/v). Ten
interferograms were averaged to produce each infrared spectrum. The
Wine Scan was configured to acquire duplicate infrared spectra for each
sample. Wave number (cm− 1) = 3.858 × pin number. The experiment
was carried out in triplicate (n = 3). The pH was measured with a digital
pH meter. The number of scans generated by each sample, selection of
wavenumber, processing of the spectrum, and statistical data analysis
settings were determined by the manufacturer and could not be changed
by the user.
2.3. Volatile compound analysis
The volatile compounds were extracted by headspace solid-phase
microextraction (HS-SPME) with divinylbenzene/carboxen/poly­
methylsiloxane (DVB/CAR/PDMS) fiber (50/30 μm, Supelco, Inc., Bel­
lefonte, PA, USA) and analyzed by gas chromatography-mass
spectrometry (GC-MS) as described by (Lu et al., 2020) with minor
modification. Subsequently, 8 mL of wine samples, 2 g of NaCl (Sino­
pharm Chemical Reagent Co., Ltd., Shanghai, China), and 10 μL of an
internal standard solution (3-octanol, 300 mg/L, Sigma-Aldrich, USA)
were held in the 20 mL headspace bottle, and the treated sample was
swirled with a vortex mixer for more than 3 s until white bubbles were
generated to mix evenly. The vials were placed in a water bath at 40 ◦ C
for 15 min for equilibration. The fiber was exposed to the sample
headspace for 40 min at 40 ◦ C and immediately followed by desorption
of the fiber in the injection port of GC-MS (7890B-5977A, Agilent, Palo
Alto, CA, USA) at 240 ◦ C for 6 min. Compounds were separated on an
HP-5MS column (60 m × 0.25 mm i.d., 0.25 μm df; J&W Science,
Folsom, CA, USA) with helium as the carrier gas at a constant flow rate
of 1 mL/min. GC was operated at the following conditions: initial tem­
perature of the column box was 40 ◦ C, increased to 80 ◦ C at a rate of
3 ◦ C/min for 6 min, and increased to 240 ◦ C at a rate of 5 ◦ C/min. MS
was operated in the electron impact ionization mode at 70 eV, and the
scanning range was 40–350 m/z. The solvent delay was set at 6.0 min to
avoid the influence of the solvent peak.
Volatile compounds were identified by comparing the MS fragmen­
tation patterns, which were obtained from the NIST 14 database with
similarity more than 80%. All the volatile compounds were quantified
by semi-quantitative analysis, and the ratio of the peak area of each
compound to the peak area of the internal standard (3-octanol) was used
to calculate the relative content of volatile compounds. Volatile com­
pound analysis was performed in triplicate (n = 3).
2.4. Organic acid analysis
Organic acids of the fermentation liquid were determined by high-
performance liquid chromatography (HPLC) as described by Castellari
et al. (2000) with minor modification. An appropriate amount of wine
fermentation liquid was centrifuged at 6201×g (8000 rpm) at 4 ◦ C for
10 min and filtered by a 0.45 μm organic phase needle filter. Finally, the
liquid was passed through a DIKMA ProElut C18 (1000 mg/6 mL of
30/pk, DIKMA, Beijing, China) activated with methanol (LiChrosolv®,
Supelco, Germany) for 1 h and collected into a 1.5 mL vial. The pro­
cessed sample was automatically injected into the HPLC (Waters 2489
UV/Visible Detector, Waters 1525 Binary HPLC Pump, Waters 2707
Autosampler). Chromatographic conditions were as follows: mobile
phase, KH2PO4 (0.02 mol/L, pH 2.4); column, Diamonsil Plus C18-A (4.6
mm × 250 mm, 5 μm, DIKMA, Beijing, China); detection wavelength,
210 nm; injection volume, 10 μL; flow rate, 0.8 mL/min; and column
temperature, 30 ◦ C. External standard was used for quantitative
X. Wang et al. Food Bioscience 55 (2023) 102952

analysis. The experiment was carried out in triplicate (n = 3).
2.5. Sensory evaluation
The wine was evaluated by ten trained assessors (6 females and 4
males, range = 25–55 years, average 35 years) from a winery and with
rich experience in Longyan wine brewing and wine tasting. Subse­
quently, 20 mL aliquots of the wines were poured into wine glasses and
presented in random order at 20 ◦ C. Potable water was provided to rinse
the palate during testing. Sensory descriptions included appearance,
green fruit, tropical fruit, stone fruit, citrus fruit, floral, aroma intensity,
mouthfeel, harmonious and typicality. The wine samples were analyzed
with a nonstructured scale of 5 cm, in which a score of 0 indicated “not
perceptible” and a score of 5 indicated “strongly perceptible.” The re­
sults are expressed as average values.
2.6. Statistical analysis
One-way ANOVA and Duncan test were completed by SPSS 26
software (SPSS Inc., Chicago, IL, USA), and the significance level was set
to P < 0.05. Data and charts were prepared by Origin 2021 (OriginLab
Corporation, Northampton, MA, USA). Principal component analysis
(PCA) was performed to identify the most influential volatile com­
pounds in different wine samples by SIMCA 14.1 software (Umetrics AB,
Umeå, Sweden). Hierarchical clustering and heat map visualization of
volatile compounds were generated in Origin 2021.
3. Results and discussion
3.1. Oenological parameters during fermentation
The oenological parameters of Longyan must samples at different
fermentation stages are shown in Table 1. The reducing sugar contents
of five different treatment wine samples at the end of fermentation were
all lower than 4 g/L, which met the requirement of dry wine. Pure
fermentation of S. cerevisiae VL2 (2.25 ± 0.13 g/L) and the sequential
fermentation of R. mucilaginosa HL-S-8/VL2 (2.47 ± 0.10 g/L) contained
significantly lower reducing sugar than other treatment samples. The
ethanol concentration of pure fermentation (11.12 ± 0.04%, v/v) was
the highest among all fermentations; thus the use of non-Saccharomyces
yeasts in fermentation with S. cerevisiae could reduce the ethanol con­
centration without fermentation, which was consistent with previous
findings reported by Aplin et al. (2019). The total acid increased from
8.6 g/L to 9.3–9.6 g/L at an early stage and then decreased to 8.3–8.5
g/L until the end of fermentation; meanwhile, the total acid content of
pure fermentation of S. cerevisiae VL2 was the lowest (8.35 ± 0.03 g/L).
The pH did not change obviously during fermentation, and it fluctuated
between 3.2 and 3.4.
3.2. Volatile compounds of wine samples in different fermentations
As shown in Table 2, 46 volatile compounds, including 26 esters, 10
alcohols, 5 acids, 4 aldehydes and ketones, and 1 other compound, were
identified from wine samples of different fermentations. Compared with
the three other fermentation treatments, sequential fermentation of
R. mucilaginosa HL-S-8/VL2 and P. kluyveri XL-48-6/VL2 significantly
increased the concentration of volatile compounds (9929.35 ± 96.87
and 9017.67 ± 85.91 μg/L, respectively).
3.2.1. Volatile compound analysis
Esters are the largest group of compounds that can provide the fruity
aroma characteristics desired in wine during fermentation, especially
acetate and ethyl esters (Aplin et al., 2019). As shown in Table 2, a total
of 26 esters were detected and classified into acetate esters, fatty acid
ethyl esters, and other esters. Fig. 1A shows that the ester contents of
fermentations of R. mucilaginosa HL-S-8/VL2 (7461.40 ± 81.58 μg/L)
and P. kluyveri XL-48-6/VL2 (6774.77 ± 131.33 μg/L) were higher than
those of pure fermentation (5528.13 ± 441.57 μg/L). This result indi­
cated that the sequential fermentations of these two non-Saccharomyces
yeasts and S. cerevisiae contributed to the formation of esters, which was
also reported by other researchers with other non-Saccharomyces yeast
species (Carrau et al., 2008; Tristezza et al., 2016; Zhang et al., 2018).
Compared with other fermentations, the sequential fermentation of
R. mucilaginosa HL-S-8/VL2 significantly increased the concentration of
volatile compounds. In particular, the contents of phenylethyl acetate
(apple, cherry, pear, and floral), ethyl octanoate (fruity, pear, apricot,
and banana), and ethyl 9-decenoate (rose and fruity) were significantly
elevated. Furthermore, the contents of hexyl acetate (apple, cherry,
pear, and floral), ethyl dodecanoate (floral, fruity, and cream), and ethyl
decanoate (fruity, pleasant, and wax) increased in the sequential
fermentation of P. kluyveri XL-48-6/VL2. These esters will bring pleasant
floral and fruity aromas to wine, which was consistent with previous
reports (Lan et al., 2022).
Alcohols, the second major group of volatile compounds, are
responsible for specific alcoholic and fruity aromas of wine (Zhao et al.,
2017). As shown in Table 2, alcohols were mainly composed of phe­
nylethyl alcohol (flowery, pollen, and perfume), isobutyl alcohol
(alcohol and mild sweet), and isoamyl alcohol (cheese, whisky, and ripe

Table 1
Changes in oenological parameters during Longyan wine different fermentations.
Parameters Fermentation time VL2 CV RV HV PV

Reducing Sugar (g/L) Early Stage 124.86 ± 1.71d 136.65 ± 1.05c 145.00 ± 2.66a 141.39 ± 3.47ab 140.26 ± 0.79bc
Middle Stage 88.29 ± 2.79bc 90.19 ± 2.15b 95.90 ± 2.75a 85.20 ± 1.65c 84.28 ± 1.26c
Late Stage 14.36 ± 0.86d 25.01 ± 1.43b 24.46 ± 0.94b 27.53 ± 1.26a 21.28 ± 0.61c
End Stage 2.25 ± 0.13c 3.70 ± 0.14a 2.47 ± 0.10c 3.48 ± 0.27ab 3.27 ± 0.08b
Ethanol (%, v/v) Early Stage 1.65 ± 0.13a 0.82 ± 0.04b 0.31 ± 0.05d 0.35 ± 0.01cd 0.46 ± 0.03c
Middle Stage 3.03 ± 0.67c 2.26 ± 0.28d 2.21 ± 0.21d 3.80 ± 0.33b 4.58 ± 0.18a
Late Stage 10.55 ± 0.26a 9.98 ± 0.11bc 9.99 ± 0.03bc 9.87 ± 0.15c 10.24 ± 0.05b
End Stage 11.12 ± 0.04a 10.88 ± 0.10b 10.95 ± 0.10b 10.90 ± 0.04b 10.92 ± 0.05b
Total Acid (g/L) Early Stage 9.35 ± 0.02c 9.43 ± 0.03b 9.37 ± 0.05c 9.59 ± 0.03a 9.56 ± 0.03a
Middle Stage 9.27 ± 0.02d 9.38 ± 0.03ab 9.33 ± 0.03c 9.42 ± 0.03a 9.34 ± 0.02bc
Late Stage 8.54 ± 0.03c 8.64 ± 0.02a 8.48 ± 0.04d 8.61 ± 0.03ab 8.57 ± 0.02bc
End Stage 8.35 ± 0.03c 8.49 ± 0.02a 8.41 ± 0.02b 8.46 ± 0.03a 8.42 ± 0.04b
pH Early Stage 3.40 ± 0.05a 3.27 ± 0.02bc 3.31 ± 0.02b 3.23 ± 0.01c 3.26 ± 0.01c
Middle Stage 3.33 ± 0.02a 3.25 ± 0.02bc 3.27 ± 0.01b 3.22 ± 0.02c 3.25 ± 0.02bc
Late Stage 3.29 ± 0.05ab 3.25 ± 0.05b 3.31 ± 0.03a 3.27 ± 0.02ab 3.27 ± 0.03ab
End Stage 3.35 ± 0.01a 3.21 ± 0.03d 3.30 ± 0.03b 3.24 ± 0.01c 3.25 ± 0.02c

Values shown represent averages of triplicate samples (data are mean ± SD). Values with different superscript roman letters in the same row are significantly different
according to the Duncan test (P < 0.05).
VL2: pure fermentation of S. cerevisiae VL2; CV: sequential fermentation of Candida stellimalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula mucilaginosa
HL-S-8/VL2; HV: sequential fermentation of Hanseniaspora uvarum HL-S-9/VL2; PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2.

3
X. Wang et al. Food Bioscience 55 (2023) 102952

Table 2
Identification and relative contents of volatile compounds of wine samples in different fermentations in Longyan wine.
Numbered Compounds Concentration (μg/L) Odor threshold Odors
(μg/L)
VL2 CV RV HV PV

∑ (46) 7654.64 ± 6738.71 ± 9929.35 ± 6950.55 ± 9017.67 ±

397.07c 432.50d 96.87a 409.70cd 85.91b
Esters (26) 5528.13 ± 4865.01 ± 7461.40 ± 5192.23 ± 6774.77 ±
441.57c 398.35d 81.58a 445.84cd 131.33b
Acetic esters (6) 437.00 ± 301.73 ± 512.11 ± 274.46 ± 458.86 ±
22.21b 27.97c 12.92a 13.65c 5.22b
[A]
V1 Phenylethyl acetate 40.99 ± 35.28 ± 54.93 ± 39.21 ± 44.35 ± 250 Apple, cherry, pear, floral,
0.66bc 2.60d 2.96a 1.06cd 1.57b pleasant [B]
V2 Heptyl acetate 10.36 ± 0.70c 8.70 ± 1.19c 15.55 ± 15.31 ± 1.56a 13.22 ± 1400 [C]
Almond, pear [B]
0.78a 0.30b
V3 Octyl acetate Nd Nd Nd 3.96 ± 0.70a Nd 50000 [C]
Green, earthy, mushroom,
herbal, waxy [B]
[A]
V4 Isoamyl acetate 315.22 ± 197.43 ± 339.51 ± 122.03 ± 289.47 ± 30 Fruity, sweet, banana [B]
21.39ab 22.87c 8.52a 12.25d 5.34b
V5 Hexyl acetate 70.44 ± 3.85c 60.32 ± 7.80c 102.11 ± 91.35 ± 1.40b 110.01 ± 1500 [A] Apple, cherry, pear, floral [B]
4.47ab 9.51a
V6 (Z)-3-Hexenyl acetate Nd Nd Nd 2.60 ± 0.34a 1.80 ± 0.29a
Fatty acid ethyl 5543.74 ± 4541.07 ± 6893.64 ± 4806.92 ± 6254.76 ±
esters (13) 333.87c 297.84c 56.67a 346.05c 105.21b
[A]
V7 Ethyl butyrate 45.58 ± 61.27 ± 69.55 ± 54.42 ± 62.15 ± 20 Pineapple, yellow passion
15.68b 5.44ab 3.45a 8.25ab 6.35ab fruit [B]
V8 Ethyl heptanoate 21.42 ± 24.48 ± 3.30a 18.34 ± 12.76 ± 1.58c 25.36 ± 0.71a 220 [A]
Pineapple [A]
1.23ab 2.31b
V9 Ethyl octanoate 2394.85 ± 1886.67 ± 2986.74 ± 2073.77 ± 2604.06 ± 2 [A] Sweet, fruity, pear, apricot,
93.23c 113.19d 68.96a 188.99d 41.39b banana [B]
[A]
V10 Ethyl dodecanoate 206.99 ± 223.07 ± 285.89 ± 163.60 ± 307.96 ± 800 Sweet, floral, fruity, cream [B]
3.31b 28.50b 15.80a 30.44b 28.38a
[A]
V11 Ethyl decanoate 858.49 ± 730.01 ± 1031.79 ± 727.11 ± 1044.98 ± 200 Fruity, fatty, pleasant, wax
116.59b 17.34b 37.69a 105.26b 31.43a flavor [B]
V12 Ethyl nonanoate 9.48 ± 0.44a 8.31 ± 1.34ab 8.45 ± 0.26ab 7.64 ± 1.20b 8.80 ± 0.55ab 1300 [A]
Floral, fruity [B]
V13 Ethyl hexanoate 577.38 ± 568.60 ± 755.17 ± 575.25 ± 736.92 ± 5 [A] Fruity, green apple, brandy,
33.40b 72.88b 32.15a 46.54b 7.96a wine-like [B]
V14 Ethyl tetradecanoate 6.73 ± 0.21c 8.40 ± 1.20b 6.84 ± 0.38c 5.21 ± 0.58d 11.90 ± 0.69a
V15 Ethyl hexadecanoate 14.06 ± 0.49c 19.06 ± 15.93 ± 16.36 ± 23.50 ± 1.59a
2.77b 0.52bc 2.05bc
V16 Ethyl 2-hexenoate 4.68 ± 0.20b 4.96 ± 0.60b 5.89 ± 0.33a 5.53 ± 0.60ab 6.09 ± 0.22a
V17 Ethyl 7-octenoate 17.08 ± 14.05 ± 2.02c 22.72 ± 15.33 ± 21.34 ± 0.60a
0.68b 0.53a 1.20bc
[A] [B]
V18 Ethyl 9-decenoate 1387.01 ± 992.19 ± 1705.33 ± 1143.07 ± 1388.13 ± 100 Rose, green, fruity, fatty
141.52b 133.31c 53.56a 55.23c 22.14b
V19 Ethyl trans-4- Nd Nd Nd 6.88 ± 1.60a 6.58 ± 0.55a
decenoate
Other esters (7) 47.38 ± 22.21 ± 55.65 ± 32.78 ± 4.68c 61.15 ± 2.90a
5.26b 0.68d 1.61a
V20 Methyl decanoate 3.11 ± 0.51a 3.14 ± 0.03a 2.82 ± 0.09ab 1.79 ± 0.17c 2.49 ± 0.12b 1.2 [C] Wine, fruity, floral [C]
V21 Methyl octanoate 14.44 ± 1.47a Nd 13.46 ± 10.71 ± 2.65b 11.93 ± 100–400 [F]
Waxy, apple skin, fruity [F]
0.72ab 1.29ab
V22 Diethyl succinate Nd Nd 4.34 ± 0.04a Nd Nd 6000 [F]
Light fruity; wine [F]

V23 Pentyl propanoate Nd Nd 2.50 ± 0.18a 2.63 ± 0.51a Nd

V24 Isoamyl octanoate 18.62 ± 19.08 ± 23.81 ± 17.65 ± 2.57b 24.68 ± 1.99a 125 [A]
Pungent, fruity, cheese [C]

2.39b 0.66b 1.68a

V25 Isoamyl hexanoate Nd Nd 8.72 ± 0.34a Nd 8.30 ± 0.50a 1000 [C] Sweet fruity, banana, apple,
pineapple, green [B]
V26 Pentyl decanoate 11.21 ± 2.10a Nd Nd Nd 13.75 ± 1.47a
Alcohols (10) 1505.76 ± 1396.88 ± 1699.18 ± 1358.29 ± 1557.17 ±
13.83bc 97.57c 72.30a 16.80d 36.89b
[D]
V27 Phenethyl alcohol 176.96 ± 246.85 ± 351.61 ± 287.23 ± 309.90 ± 14000 Flowery, pollen, perfume [D]
14.41d 14.76c 10.96a 11.26b 24.67b
V28 1-Propanol 36.42 ± 1.47a Nd Nd Nd Nd 306000 [E] Ripe fruit, alcohol [E]
V29 Isobutyl alcohol 45.86 ± 41.07 ± 3.30c 84.75 ± 61.08 ± 56.57 ± 40000 [F] Alcohol, mild sweet [B]
0.78bc 3.06a 13.95b 12.73bc
V30 Citronellol 12.18 ± 2.42a 8.08 ± 2.27b 11.20 ± 7.19 ± 0.58b 7.79 ± 0.22b 100 [A]
Green lemon [A]

0.91a
V31 1-Hexanol 107.89 ± 87.74 ± 105.60 ± 99.09 ± 107.49 ± 8000 [A] Green, herb [A]

1.35a 7.97b 2.30a 7.36ab 8.28a

V32 1-Heptanol 46.51 ± 1.03a 38.48 ± 42.26 ± 46.02 ± 6.42a 40.66 ± 1000 [C] Grape, sweet [C]

3.38b 0.87ab 1.68ab

[A]
V33 Isoamyl alcohol 1054.52 ± 970.48 ± 1076.36 ± 853.84 ± 1011.60 ± 30000 Cheese, whisky, ripe fruit [C]
9.64a 88.14a 103.15a 16.27b 22.89a
V34 (E)-3-Hexen-1-ol 3.60 ± 0.53a 3.17 ± 0.27a 3.62 ± 0.19a 3.84 ± 0.19a 4.17 ± 0.92a 400 [C]
Green, herb [B]

V35 3-Cyclohexen-1-ol 21.83 ± 0.52a Nd 21.21 ± Nd 18.99 ±

0.60a 0.99b
(continued on next page)

4
X. Wang et al. Food Bioscience 55 (2023) 102952

Table 2 (continued )
Numbered Compounds Concentration (μg/L) Odor threshold Odors
(μg/L)
VL2 CV RV HV PV

V36 (S)-3-Methyl-1- Nd Nd 2.37 ± 0.07a Nd Nd 1000 [C]

Chemical fusel [C]
pentanol
Acid (5) 600.49 ± 454.25 ± 742.72 ± 370.87 ± 665.12 ±
24.63b 18.41c 40.26a 30.76d 42.98b
V37 Acetic acid 15.85 ± 22.94 ± 2.85a 17.07 ± 27.67 ± 0.48a 22.40 ± 3.13a 200000 [C]
Acid, fatty [C]

3.17b 1.87b
[A]
V38 Octanoic acid 339.16 ± 260.87 ± 423.12 ± 219.39 ± 379.49 ± 500 Rancid, harsh, cheese, fatty
14.51b 14.40c 20.39a 22.22c 25.37b acid [H]
V39 Hexanoic acid 74.98 ± 68.02 ± 96.45 ± 58.35 ± 2.81c 88.00 ± 420 [A]
Cheese, rancid [H]
4.75b 4.51bc 3.51a 10.69a
V40 n-Decanoic acid 108.24 ± 76.91 ± 124.95 ± 49.12 ± 3.93c 118.73 ± 15000 [C]
Fatty, rancid [B]

8.17a 2.58b 12.36a 7.12a

V41 9-Decenoic acid 62.26 ± 25.51 ± 1.53c 72.12 ± 16.36 ± 2.97d 56.50 ±
2.29b 4.26a 2.93b
Aldehydes and 11.01 ± 16.70 ± 1.98a 11.61 ± 18.72 ± 4.50a 11.35 ±
ketones (4) 0.84b 0.68b 0.25b
V42 Acetaldehyde Nd 5.57 ± 2.67a Nd 6.83 ± 4.80a Nd 110000 [E]
Pungent, ripe apple [E]
V43 3-Octanone 6.24 ± 0.47c 6.98 ± 0.19ab 4.13 ± 0.21d 7.27 ± 0.42a 6.33 ± 0.24bc 21.4 [C] Herbal [C]
V44 Damascenone Nd Nd Nd 4.62 ± 0.33a Nd
V45 β-Damascenone 4.77 ± 0.65b 4.14 ± 0.32b 7.48 ± 0.58a Nd 5.02 ± 0.18b 0.14 [I]
Honeyed apple, dry plum,
fruity [B]
Others (1) 9.26 ± 2.27b 6.87 ± 1.08b 14.45 ± 10.44 ± 9.26 ± 0.88b
4.24a 1.66ab
V46 2,4-Di-tert- 9.26 ± 2.27b 6.87 ± 1.08b 14.45 ± 10.44 ± 9.26 ± 0.88b 200 [J]
Roses, floral [J]

butylphenol 4.24a 1.66ab

Values shown represent averages of triplicate samples (data are mean ± SD). Values with different superscript roman letters in the same row are significantly different
according to the Duncan test (P < 0.05). Nd means the compound was not detected by GC-MS in the corresponding wine sample.
VL2: pure fermentation of S. cerevisiae VL2; CV: sequential fermentation of Candida stellimalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula mucilaginosa
HL-S-8/VL2; HV: sequential fermentation of Hanseniaspora uvarum HL-S-9/VL2; PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2. Odor threshold and odors
were obtained from literatures: [A] (Shi et al., 2019); [B] (Shi et al., 2022); [C] (Chen et al., 2022); [D] (Zhu et al., 2021); [E] (Peinado et al., 2006); [F] (Peng et al.,
2013); [G] (Hu et al., 2016); [H] (Tao & Li, 2009); [I] (Lu et al., 2020); [J] (Welke et al., 2022).

Fig. 1. Four series of aroma compounds content of

wine samples in different fermentations. A. Esters
content; B. Alcohols content; C. Acids content; D.
Aldehydes and ketones content Values with different
superscript roman letters in the same row are signif­
icantly different according to the Duncan test (P <
0.05) VL2: pure fermentation of S. cerevisiae VL2; CV:
sequential fermentation of Candida stellimalicola HL-
S-5/VL2; RV: sequential fermentation of Rhodotorula
mucilaginosa HL-S-8/VL2; HV: sequential fermenta­
tion of Hanseniaspora uvarum HL-S-9/VL2; PV:
sequential fermentation of Pichia kluyveri XL-48-6/
VL2.

fruit) in all fermentation methods. As shown in Fig. 1B, when alcoholic
fermentation finished, the total alcohol concentration of the sequential
fermentation of R. mucilaginosa HL-S-8/VL2 was the highest (1699.18 ±
72.30 μg/L), followed by that of the sequential fermentation of
P. kluyveri XL-48-6/VL2 (1557.17 ± 36.89 μg/L). In particular, the
content of phenethyl alcohol and isobutyl alcohol in the sequential
fermentation of R. mucilaginosa HL-S-8/VL2 was significantly higher
than that in the other samples in the same period. Phenylethyl alcohol
has been reported as a potential factor in the production of floral aromas
in wine (de-la-Fuente-Blanco et al., 2016), and wines containing high
levels of isobutyl alcohol and isoamyl alcohol are superior wines (Xie
et al., 2016). Furthermore, citronellol was significantly abundant in the
sequential fermentation of R. mucilaginosa HL-S-8/VL2 and pure
fermentation of S. cerevisiae, which may bring the aroma of green lemon
to the wine. These results indicated that the fermentation of
R. mucilaginosa HL-S-8/VL2 increased the aroma concentration and

5
X. Wang et al.
complexity.
Acid is considered an important factor in the balance of aromas and
flavors of wine, and it is produced through the metabolism of yeast and
lactic acid bacteria. As shown in Table 2 and Fig. 1C, the production of
fatty acids was higher in the sequential fermentation of R. mucilaginosa
HL-S-8/VL2 (742.72 ± 40.26 μg/L) than in the other samples in the
same period. However, these five substances could not contribute
negatively to the aroma of wines due to their high olfactory threshold.
Excessive fatty acids in wine have been described as having a cheesy,
fatty, and putrid taste (Bisson & Karpel, 2010), whereas fatty acids are
necessary substrates for the synthesis of fatty acid ethyl esters (Andorra
et al., 2010). Therefore, the sequential fermentation of R. mucilaginosa
HL-S-8/VL2 would contribute to the formation of fatty acid in wine.
Aldehydes, the main source of herbaceous compounds in wine, pri­
marily include benzaldehyde. They mainly come from the oxidation of
fatty acids and degradation of amino acids caused by microbial
fermentation (Zhao et al., 2020). As shown in Table 2, the production of
β-damascenone was higher in the sequential fermentation of
R. mucilaginosa HL-S-8/VL2 (7.48 ± 0.58 μg/L) than in the other sam­
ples. Despite its relatively low concentrations, β-damascenone positively
contributed to the wine aroma’s honeyed apple, dry plum, and fruity
notes because of its low odor threshold. Overall, the sequential
fermentation of R. mucilaginosa HL-S-8/VL2 increased the aromatic and
balanced sensory characteristics of wine.
3.2.2. PCA of wine aroma components in different fermentations
PCA was carried out to reveal the correlation and segregation of
various aroma compounds in different wine samples (Fig. 2). Here,
75.3% of the variance was explained by all components detected at the
end of fermentation, and PC1 and PC2 accounted for 50.6% and 24.7%
of the variance, respectively. The PCA results indicated that the
sequential fermentation of P. kluyveri XL-48-6/VL2 was grouped in the
upper right quadrant with isobutyl alcohol and some esters. The
sequential fermentation of R. mucilaginosa HL-S-8/VL2 was located in
the lower right quadrant with multiple aroma compounds, such as fatty
acid ethyl esters and acid, contributing to the improvement of floral and
fruity flavors, and it was conducive to the balance of aromas and flavors
of wine. The sequential fermentation of H. uvarum HL-S-9/VL2 was
grouped in the upper left quadrant with acetic acid, representing slight
Fig. 2. Bioplot of PCA for volatile compound of wine samples in different
fermentations. : Wine samples; : Esters; : Alcohols; : Acid VL2: pure
fermentation of S. cerevisiae VL2; CV: sequential fermentation of Candida stel­
limalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula mucilaginosa
HL-S-8/VL2; HV: sequential fermentation of Hanseniaspora uvarum HL-S-9/VL2;
PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2.
6
Food Bioscience 55 (2023) 102952
rancidity. Furthermore, the sequential fermentation of pure fermenta­
tion of S. cerevisiae VL2 was located in the lower left quadrant with 3-
cyclohexen-1-ol and methyl decanoate (fruity and floral). However,
the sequential fermentation of C. stellimalicola HL-S-5/VL2 was grouped
in the lower left quadrant with low amounts of compounds. As shown in
Fig. 2, these five samples were relatively far away from one another in
the plot, indicating a difference in volatile aromas. These results illus­
trated that R. mucilaginosa HL-S-8 could be used to sequentially ferment
with S. cerevisiae VL2 to produce wine with more kinds of flavor
compounds.
3.2.3. Hierarchical cluster analysis (HCA) of all aroma compounds in
different fermentations
The dendrogram of HCA was applied to visualize the differences in
aroma compounds among different fermentations (Fig. 3). The results
showed that the aroma compound composition of the sequential
fermentation of C. stellimalicola HL-S-5/VL2 was closer to that of
S. cerevisiae pure fermentation. The sequential fermentation of
R. mucilaginosa HL-S-8/VL2 and P. kluyveri XL-48-6/VL2 showed no
typical aroma profile differences. Moreover, the aroma compounds were
divided into four classes. Class I mainly included partial esters, alde­
hydes, and ketones. Class II mainly contained fatty acid ethyl esters.
Class III mainly included alcohol and other esters. Class IV mainly
contained esters, alcohols, and acids. As shown in Fig. 3, the sequential
fermentation of H. uvarum HL-S-9/VL2 was rich in class I compounds,
sequential fermentation of P. kluyveri XL-48-6/VL2 was abundant in
class II compounds, pure fermentation of S. cerevisiae VL2 was highly
enriched with Class III compounds, and sequential fermentation of
R. mucilaginosa HL-S-8/VL2 was highly populated with Class IV com­
pounds. The fermentation of P. kluyveri XL-48-6/VL2 was rich in some
fatty acid ethyl esters. The high content of acetic esters and fatty acid
ethyl esters resulted in the fruity flavor of wine, and excess content of
alcohols and fatty acids would present unpleasant green, pungent and
rancid flavor in the wine, respectively (Ciani et al., 2010; Swiegers &
Pretorius, 2005). Consequently, the sequential fermentation of
R. mucilaginosa HL-S-8/VL2 and P. kluyveri XL-48-6/VL2 might produce
wines with more harmonious wine aroma than other fermentations.
3.3. Organic acids of wine samples in different fermentations
Organic acids have been reported to be an important factor influ­
encing aroma, taste, and microbiological stability of wine (Wei et al.,
2019). Significant differences were noted in the organic acid concen­
tration and composition among the wine samples in different fermen­
tations (Table 3). Tartaric and lactic acid were the primary organic acids
in dry white wine, which was consistent with a previous report (Wang
et al., 2015). Compared with the must sample, the concentration of
lactic and succinic acid in wine samples fermented with different
non-Saccharomyces yeast strains increased, whereas malic, tartaric, and
citric acid decreased during fermentation.
The sequential fermentation of R. mucilaginosa HL-S-8/VL2 had the
highest tartaric (1633.89 ± 31.73 mg/L) and lactic acid (939.45 ±
37.15 mg/L) contents, followed by the pure fermentation of S. cerevisiae
VL2 (1596.74 ± 8.84 and 870.97 ± 21.07 mg/L, respectively). Tartaric
acid is a dominant organic acid in wine, which plays an important role in
grape ripening and wine fermentation. Lactic acid improves the stability
and nutritional value of fermented juice and reduces the sharp taste
(Peng et al., 2021) and increases the complexity (buttery, spiced, roas­
ted, vanilla, and smoked notes) and fruit aroma of wine (Cappello et al.,
2017). Among them, the contents of succinic acid (437.44 ± 8.15 mg/L)
and malic acid (169.35 ± 7.50 mg/L) in the wine fermented by
P. kluyveri XL-48-6/VL2 were the lowest, followed by those of the
fermentation of H. uvarum HL-S-9/VL2 (537.00 ± 28.19 and 201.39 ±
2.47 mg/L, respectively). Succinic acid is created as a by-product of the
wine fermentation process of sugar. This acid makes the wine taste salty,
bitter, and acidic (Kritsunankul et al., 2009). Malic acid provides a
X. Wang et al. Food Bioscience 55 (2023) 102952

Fig. 3. Hierarchical clustering and heat map visualization of volatile compounds of wine samples in different fermentations. VL2: pure fermentation of S. cerevisiae
VL2; CV: sequential fermentation of Candida stellimalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula mucilaginosa HL-S-8/VL2; HV: sequential
fermentation of Hanseniaspora uvarum HL-S-9/VL2; PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2.

Table 3
Content of organic acids of wine samples in different fermentations.
Organic acids Concentration (mg/L)

Must VL2 CV RV HV PV
a c f b d
Tartaric acid 2096.94 ± 4.06 1596.74 ± 8.84 1085.25 ± 5.15 1633.89 ± 31.73 1423.96 ± 5.16 1354.19 ± 3.27e
Lactic acid 123.67 ± 10.53e 870.97 ± 21.07b 664.85 ± 45.21d 939.45 ± 37.15a 834.97 ± 20.96b 745.37 ± 4.62c
Succinic acid 33.67 ± 8.66d 580.28 ± 3.99b 692.10 ± 66.76a 679.22 ± 35.32a 537.00 ± 28.19b 437.44 ± 8.15c
Citric acid 444.32 ± 34.38a 375.98 ± 9.36b 382.76 ± 1.16b 373.34 ± 17.14b 368.72 ± 10.93bc 331.81 ± 10.05c
Malic acid 237.72 ± 4.18a 223.10 ± 7.65ab 225.39 ± 14.25ab 217.42 ± 8.68bc 201.39 ± 2.74c 169.35 ± 7.50d
Total acid 2936.31 ± 25.70d 3647.07 ± 39.91b 3050.33 ± 95.26d 3843.32 ± 73.93a 3366.04 ± 59.73c 3038.16 ± 18.72d

Values with different superscript roman letters in the same row are significantly different according to the Duncan test (P < 0.05).
VL2: pure fermentation of S. cerevisiae VL2; CV: sequential fermentation of Candida stellimalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula mucilaginosa
HL-S-8/VL2; HV: sequential fermentation of Hanseniaspora uvarum HL-S-9/VL2; PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2.

strong and sharp taste sensation, but excess levels can result in a pungent organic acid contents than the other samples, and this result indicated
taste (Zhang et al., 2008). Thus, compared with the wine fermented by that R. mucilaginosa HL-S-8 increased mouthfeel richness and
commercial S. cerevisiae VL2 strain, the sequential fermentation of complexity.
R. mucilaginosa HL-S-8/VL2 could significantly increase the contents of
lactic acid. Meanwhile, the contents of malic acid could be significantly
3.4. Sensory analysis
reduced by fermentation of H. uvarum HL-S-9/VL2 and P. kluyveri
XL-48-6/VL2. Except for the lower citric acid content of the fermenta­
As shown in Fig. 4, the results of the sensory evaluation of different
tion of P. kluyveri XL-48-6/VL2 (331.81 ± 10.05 mg/L), the citric acid
wine samples indicated that the sequential fermentation of
contents were quite similar among the wines. Citric acid is a kind of
R. mucilaginosa HL-S-8/VL2 and P. kluyveri XL-48-6/VL2 obtained a
natural fruit acid with strong acidity. As shown in Table 3, the sequential
higher score in green fruit, tropical fruit, aroma intensity and floral notes
fermentation of R. mucilaginosa HL-S-8/VL2 had significantly higher
than S. cerevisiae pure fermentation. The sequential fermentation of

7
X. Wang et al.
Fig. 4. Sensory descriptive analysis of wine samples in different fermentations.
VL2: pure fermentation of S. cerevisiae VL2; CV: sequential fermentation of
Candida stellimalicola HL-S-5/VL2; RV: sequential fermentation of Rhodotorula
mucilaginosa HL-S-8/VL2; HV: sequential fermentation of Hanseniaspora uvarum
HL-S-9/VL2; PV: sequential fermentation of Pichia kluyveri XL-48-6/VL2.
R. mucilaginosa HL-S-8/VL2 obtained the highest score, followed by the
sequential fermentation of P. kluyveri XL-48-6/VL2, pure fermentation of
S. cerevisiae VL2, sequential fermentation of H. uvarum HL-S-9/VL2 and
C. stellimalicola HL-S-5/VL2. The differences in appearance and typi­
cality were not significant in different wine samples. There was a dif­
ference in the scores of citrus fruity and aroma intensity, which may be
caused by the content of ethyl butyrate (Feng et al., 2018). Moreover,
wines obtained from the sequential fermentation of R. mucilaginosa
HL-S-8/VL2 stood out for obtaining higher scores in mouthfeel, followed
by pure fermentation of S. cerevisiae VL2. Thus, the R. mucilaginosa
HL-S-8 and P. kluyveri XL-48-6 strains were suitable to ferment wine
with better flavor and quality than the other strains, and sequential
fermentation of these two strains and S. cerevisiae VL2 have great po­
tential to produce Longyan wine.
4. Conclusions
This work investigated the effect of the sequential fermentation of
four indigenous non-Saccharomyces yeasts and S. cerevisiae VL2 on the
physicochemical properties, volatile compositions, organic acids, and
sensory characteristics of Longyan dry white wine. All non-Saccharo­
myces yeasts could generate Longyan wine with the correct levels of
alcohol, total sugar, and total acid. A total of 46 volatile compounds
were detected in all fermentations, and esters and alcohols were the
dominant groups. Compared with pure fermentation, the sequential
fermentations of R. mucilaginosa HL-S-8/VL2 and P. kluyveri XL-48-6/
VL2 increased the concentration of total volatile compounds. In partic­
ular, the sequential fermentation of R. mucilaginosa HL-S-8/VL2 pro­
duced wines with a high content of fermentative aromas, such as acetic
esters and fatty acid ethyl esters, which enhanced fruity and floral
characteristics. Additionally, the sequential fermentation of
R. mucilaginosa HL-S-8/VL2 had significantly higher organic acid con­
tents than the other samples and increased mouthfeel richness. The
sensory evaluation results also indicated that the sequential fermenta­
tions of R. mucilaginosa HL-S-8/VL2 obtained the highest sensory eval­
uation scores, followed by the sequential fermentation of P. kluyveri XL-
48-6/VL2.
In general, the sequential fermentation of R. mucilaginosa HL-S-8/
VL2 was effective in producing wine of high quality, and this strain
has great potential to produce Longyan wine. This work not only high­
lighted the role of these indigenous non-Saccharomyces yeast strains in
8
Food Bioscience 55 (2023) 102952
improving wine flavor and enhancing the regional characteristics, but
also provided a reference for the selection of yeast by wineries for
fermentation production of Longyan dry white wine. Furthermore, more
studies are necessary to know the fermentative behavior of these non-
Saccharomyces strains for commercial application in the future.
Author statement
Xiaodi Wang: methodology, writing-original draft, data curation.
Jiawei Chen: investigation. Xiaoxin Ge: writing-review and editing.
Xiaofang Fu: data curation, software. Chao Dang: data curation. Jie
Wang: funding acquisition, methodology. Yaqiong Liu: conceptualiza­
tion, writing-review and editing. All authors have read and agreed to the
published version of the manuscript.
Declaration of competing interest
The authors confirm that there are no conflicts of interest in the
manuscript.
Data availability
Data will be made available on request.
Acknowledgements
This research was supported by Key Research and Huailai County
Science and Technology Support Program [grant number 2021C-05].
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