Optimization of fermentation conditions, physicochemical profile and sensory quality analysis of seedless wampee wine

Abstract

The aims of the present stud were to optimize fermentation parameters of seedless wampee wine using response surface methodology (RSM) and evaluate the changes in flavor metabolites during fermentation. Seedless wampee wine of optimal sensory quality was produced using an inoculum concentration of 0.6%, initial sugar levels of 200 g/L, a fermentation temperature of 22 °C, and a fermentation period of 9 days. Then the flavor compound profiles (amino acids, organic acids and volatile aroma compounds) of seedless wampee wine during the fermentation under optimal conditions were analyzed using high performance liquid chromatography (HPLC) and gas chromatography–mass spectrometr (GC-MS). The main fermented phase of fermentation resulted in fluctuations in both total amino acids and organic acids, with stabilization occurring later on. A total of 54 volatile components, including esters, alcohols, terpenes, and acids, were putatively identified. Terpenes were the primary drivers of the flavor characteristics of seedless wampee. The rise of esters and decline of terpenes have the potential to significantly alter the flavor of wine during fermentation. These results would contribute to the further development of seedless wampee wine.
Graphical Abstract

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Wang et al. Applied Biological Chemistry (2024) 67:81
https://doi.org/10.1186/s13765-024-00938-y
*Correspondence:
Gongliang Liu
gongliangliu@126.com
Full list of author information is available at the end of the article
Abstract
The aims of the present stud were to optimize fermentation parameters of seedless wampee wine using response
surface methodology (RSM) and evaluate the changes in avor metabolites during fermentation. Seedless
wampee wine of optimal sensory quality was produced using an inoculum concentration of 0.6%, initial sugar
levels of 200g/L, a fermentation temperature of 22°C, and a fermentation period of 9 days. Then the avor
compound proles (amino acids, organic acids and volatile aroma compounds) of seedless wampee wine during
the fermentation under optimal conditions were analyzed using high performance liquid chromatography (HPLC)
and gas chromatography–mass spectrometr (GC-MS). The main fermented phase of fermentation resulted in
uctuations in both total amino acids and organic acids, with stabilization occurring later on. A total of 54 volatile
components, including esters, alcohols, terpenes, and acids, were putatively identied. Terpenes were the primary
drivers of the avor characteristics of seedless wampee. The rise of esters and decline of terpenes have the
potential to signicantly alter the avor of wine during fermentation. These results would contribute to the further
development of seedless wampee wine.
Optimization of fermentation conditions,
physicochemical prole and sensory quality
analysis of seedless wampee wine
HongWang1,2, XiangLiao1, ChunyaoLin1, WeidongBai1,2, GengshengXiao1,2, XingyuanHuang3 and
GongliangLiu1,2*

Page 2 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
Introduction
Clausena lansium (Lour.) Skeels, commonly known as
wampee, is a fruit crop of the Rutaceae family that is
native to southern China, ailand and Vietnam [1].
Wampee fruit is renowned for its rich taste and avor,
and has been introduced to India, Sri Lanka, Australia,
the United States and in Central America [2, 3]. Previ-
ous studies have identied multiple key avor and taste
components in dierent parts of the wampee fruit, which
explained the reason that many consumers tend to eat
the pulp and peel rather than the seed of wampee [4]. As
an edible medicinal and non-medicinal fruit, wampee is
nowadays available in various forms such as fruit cups,
gelatins, juices, jams, jellies, and pies [2]. Wampee has
also been used to treat bronchitis in traditional Chinese
and Vietnamese medicine [5]. Recent studies have identi-
ed its extensive health benets, including anti-oxidation,
anti-bacterium, anti-inammation, antihypertension,
neuroprotection, and prebiotic eects, which are mainly
attributed to the bioactive phenolics, carbazole alka-
loids, and polysaccharides [6–10]. Wampee can be clas-
sied according to its taste, which can be either sweet or
sweet-sour. e sweet variety is typically consumed fresh,
while the sweet-sour variety is commonly used as a raw
material for the production of processed foods, such as
preserved fruit, wine, and vinegar [11]. Seedless wampee
(C. lansium S. cv. WuHeHuangPi), a sweet–sour cultivar,
is characterized by its thin skin, thick esh and distinc-
tive sweet-sour avor. Due to the expanded cultivation of
seedless wampee and the seasonality of the fruit, various
wampee-derived products need to be developed urgently.
Nowadays, fruit wine is gaining popularity among
consumers because of its special avor and nutritional
benets. Various types of fruit, such as grapes, apples,
durians and red pitayas, can be used for making fruit
wine [12–15]. In Vietnam, wampee is fermented with
sugar, to produce a beverage that resembles champagne
[2]. Fruit wine production not only resolves production,
marketing, transportation and preservation issues dur-
ing the fruit harvest season, but also enhances sensory
quality and physical and chemical properties. Flavor
metabolites are recognized as crucial indicators of wine
quality and play a major role in consumer purchasing
decisions [16, 17]. e composition and concentration
of primary metabolites, specially carbohydrates, organic
acids and amino acids, are closely correlated with the
avor and taste of fruit wine [18, 19]. Additionally, dur-
ing fermentation, microorganisms can break down
Graphical Abstract
Keywords Seedless wampee wine, Response surface method, Amino acid, Organic acid, Volatile compounds

Page 3 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
complex macromolecules into smaller molecules with
improved bioaccessibility and bioavailability. Enzymes
also facilitate the formation and release of avor com-
pounds to enhance the quality of fruit wine [20]. Over
the last decade, researchers have examined the poten-
tial health benets of consuming appropriate fruit wine,
especially the non-volatile components, such as phenolic
compounds and polysaccharide [21–23]. Nevertheless,
limited information is available on the dynamic avor
changes of wampee during fermentation is still unknown,
particularly for seedless wampee.
e commercial value of fruit wine depends on its
organoleptic and sensory characteristics which are
inuenced by a combination of non-volatile and volatile
components. e compositions and abundance of these
components are closely associated with the fermenta-
tion process of fruit wine. Hence, we optimized various
variables, such as inoculum concentration, initial sugar,
time, and temperature using response surface methodol-
ogy (RSM) to enhance the quality attributes of seedless
wampee wine. Additionally, we evaluated the uctuation
of physicochemical indicators, such as free amino acids,
organic acids and volatile aroma compounds. Hopefully,
these results would provide a scientic basis for seed-
less wampee wine production and increase the versatile
application of seedless wampee in the food industry.
Materials and methods
Materials and reagents
Seedless wampee was collected in Yunfu, Guangdong
Province, China. Saccharomyces cerevisiae yeast was
purchased from Angel Yeast Co., Ltd. (Hubei, China).
Lallzyme EX-V was purchased from Lallemand Inc. (Tou-
louse, France). e materials used in winemaking were
of food grade. Folin-Ciocalteu’s phenol reagent (99%),
L-ascorbic acid (99%), gallic acid (99%), protocatechuic
acid (99%), chlorogenic acid (99%), vanillic acid (99%),
syringic acid (99%), coumaric acid (99%), ferulic acid
(99%), rutin (99%), quercetin (99%), uorescein sodium
salt (99%), 2,2-azobis(2-methylpropionamidine) dihydro-
chloride (AAPH) (99%) were procured from Aladdin Ltd.
(Shanghai, China). All the chemicals and reagents used in
this study were of analytical grade.
Seedless wampee wine preparation
e fermentation process was shown in Fig. 1. Briey,
seedless wampee was washed with water, then water (1:1,
v/v) was added to crush and pulp with a homogenizer,
after which 100mg/kg SO2 was added to inhibit miscel-
laneous bacteria. en 0.1% (w/v) Lallzyme EX-V was
added and treated at 50°C for 1h for clarication, fol-
lowed by inactivation in a water bath at 80°C for 10min.
Meanwhile, 1g of yeast was inoculated into a 100 mL of
3% (v/v) sugar solution and placed in 38 °C for activa-
tion. Afterwards, the activated yeast solution (0.5–0.7%,
v/v) was inoculated into the wampee pulp, which was
adjusted for the initial sugar level (190–210 g/L) and
subjected to pre-fermentation at a suitable temperature
(21–23 °C) for 8–10 d. In order to analyze changes in
physicochemical indicators and sensory quality during
fermentation, seedless wampee was fermented under
optimal process conditions, and the upper wine layer was
collected and subjected to post-fermentation at 20 °C.
e samples were collected on day 0, 2, 3, 5, 7, 9, 14, 19,
24 and 29 for further analysis.
Response surface methodology for optimization of
fermentation conditions
In this study, Box-Behnken design and response surface
methodology (BBD-RSM) was used to investigate the
eect of fermentation factors on the quality of seedless
wampee wine [24]. e sensory score of seedless wampee
wine was taken as the response variable (Y) (Table S1).
e experimental design was applied with four inde-
pendent variable factors including inoculum size, initial
sugar, fermentation time, and fermentation temperature
at three levels, as indicated in Table1.
Sensory evaluation
Sensory evaluation of seedless wampee wine was carried
out in accordance with national standards of the PRC by
ten semi-trained panels, respectively [25]. Plain water
was provided to panelists between the evaluations of dif-
ferent samples to avoid lingering aftertaste. Scores were
given by evaluators for appearance (0–30), aroma (0–30),
taste (0–30) and typicality (0–20), respectively (Table S1).
e study was reviewed and approved by the Institutional
Fig. 1 Preparation of seedless wampee wine

Page 4 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
Review Board (IRB) of Zhongkai University of Agricul-
ture and Engineering and informed consent was obtained
from each subject prior to their participation in the study.
Amino acids analysis
Briey, concentrations of amino acids in seedless wampee
wine were analyzed by the high performance liquid chro-
matography (HPLC) system (1260, Agilent Technologies,
USA), with Advance Bio 3A amino acid chromatography
columns (4.6 × 100 mm, 2.7 μm, Agilent Technologies,
USA) [26]. e sample was centrifuged at a rate of 1000
r/min for 10min, and then the supernatant was ltered
through a 0.45μm membrane for analysis. Ten millimo-
lars Na2HPO4- Na2B4O7 (1:1, v/v) which adjust the pH to
8.2 was used as buer A and acetonitrile-methanol-acetic
acid (45:45:10, v/v) was used as buer B. e ow rate
was xed at 1 mL/min and the column temperature was
set at 40°C. e absorbance was monitored at 338nm.
e program began with 98% A and 2% B. en the buer
A linearly dropped to 43% from 0 to 13.4min, dropped
to 0% from 13.4 to 15.8min, and subsequently, rose back
to 98% from 15.8 to 20min. e chromatographic peaks
were analyzed qualitatively and quantitatively mainly by
comparison with standards.
Organic acids analysis
e method used for the determination of organic acids
was based on a previous report [27]. e HPLC system
(1260, Agilent Technologies, USA) with ZORBAX SB-Aq
columns (250 × 4.6mm, 5μm, ermo Fisher Scientic,
USA) was applied for organic acid detection. Buer A
consisted of 10% methanol and 0.01mol/L KH2PO4 solu-
tion was used as buer B. e isocratic elution program
was started with 3% A and 97% B and the absorbance was
monitored at 241nm. e other details were as follows:
injection volume was 10 µL, ow rate was 0.8 mL/min,
and the column temperature was 30 °C. Identication
and quantication were conducted using the external
standard method.
Volatile analysis
Volatile compounds were analyzed using the headspace
solid-phase microextraction-gas chromatography-mass
spectrometry (HS-SPME-GC-MS) system (7890B, Agi-
lent Technologies, USA) [28]. In brief, 7 mL of wine sam-
ples were mixed with 0.4g of NaCl in 15 mL headspace
vials. en, 10 µL of 2-octanol (2.2 mg/L) was added
as the internal standard, and the headspace vials were
sealed. Subsequently, samples were equilibrated at 45°C
for 50min, and the volatile compounds were extracted
from the headspace to the solid-phase microextraction
(SPME) ber. Separation of volatile components was per-
formed with a DB-WAX UI gas chromatography column
(30m × 250μm, 0.25 μm, Agilent Technologies, USA).
Helium at a ow rate of 1 mL/min was used as the car-
rier gas. e transfer line was set to 250°C and the ion-
source temperature was set to 230 °C. e ionization
energy of the impact was 70eV, with a scanning range of
m/z from 35 to 450. e SPME ber was placed into a gas
chromatograph injection port and desorbed for 5min at
250°C. e oven temperature was initially maintained at
40°C for 5min, and increased to 120°C at a rate of 3°C/
min and kept for 3min, followed by another increase in
temperature to 220°C at a rate of 6°C/min with a nal
holding of 5 min. e compounds were identied by
comparing their mass spectra against synthetic standards
and matches from NIST 2.0 library.
Statistical analysis
Data were expressed as means ± standard deviation
(SD, n = 3). Signicant dierences (p < 0.05) were ana-
lyzed using one-way analysis of variance (ANOVA) and
Table 1 Treatment combinations with results of the response
surface of seedless wampee wine
Run Independent variable Response
X1
Inoculum
size (%)
X2
Initial
sugar
(g/L)
X3
Fermen-
tation
time (d)
X4
Fermentation
temperature
(°C)
Y
Sensory
evaluation
(scores)
1 0.7 210 9 22 91.5
2 0.6 200 9 22 94.3
3 0.5 200 8 22 90.5
4 0.6 200 9 22 93.5
5 0.7 200 10 22 90.1
6 0.5 210 9 22 93.4
7 0.6 190 9 23 87.4
8 0.7 200 9 21 90.2
9 0.5 190 9 22 88.2
10 0.6 190 10 22 91.8
11 0.6 200 9 22 94.9
12 0.6 200 10 23 89.7
13 0.5 200 10 22 89.3
14 0.6 190 9 21 87.7
15 0.6 200 9 22 94.3
16 0.6 190 9 21 90.8
17 0.6 200 9 22 95.7
18 0.7 200 8 22 91.6
19 0.6 200 8 21 85.6
20 0.6 190 8 22 89.5
21 0.6 200 8 23 88.1
22 0.6 200 10 21 88.3
23 0.6 210 8 22 90.6
24 0.7 190 9 22 91.7
25 0.6 210 9 23 86.1
26 0.6 210 10 22 90.7
27 0.5 200 9 21 85.4
28 0.7 200 9 23 91.1
29 0.5 200 9 23 88.6

Page 5 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
Duncan’s multiple comparison post-test using SPSS
statistical software 21.0 (SPSS Inc., Chicago, IL, USA).
Design-Expert 8.0.7 was applied to establish the second-
order polynomial equation and generate the contour
plots based on analysis of variance and the optimization.
A dose-eect analysis was performed using Calcusyn
software version 2.0 (Biosoft, Cambridge, UK). Multivari-
ate data analysis was performed using SIMCA software
14.0 (Umetrics, Umeaa, Sweden). For GC-MS data, mass
spectral resolution and comparison with the NIST 20.0
standard library were used. Results with > 80% match
were retained for qualitative analysis. e relative con-
tent of each component was calculated using the internal
standard method.
Results and discussion
Eects of dierent fermentation conditions on sensory
evaluation
Sensory evaluation plays an indispensable role in the
quality of fruit wine fermentation. is study indi-
cated that optimization of fermentation conditions can
improve the sensory quality of seedless wampee wine.
e sensory scores of seedless wampee wine under dier-
ent fermentation conditions (inoculum size, initial sugar,
fermentation time, and fermentation temperature) were
shown in Table1. e second-order polynomial response
surface model tted for sensory quality was displayed in
Eq.(1):
Y
(
sensoryevaluation, scores
)=
94.54 +0.9X1+0.57X2+0.33X3+0.25X4−
1.35X1X2−0.075X1X3−0.58X1X4−0.55X2X3−
1.1X2X4−0.28X3X4−1.57X12−
1.84X2
2
−2.29X3
2
−4.39X4
2
(1)
e results of the analysis of variance for the regression
model using RSM were presented in Table2. e regres-
sion model was indicated to be signicant with a p- value
of 0.0016 (p < 0.01). e quadratic polynomial model for
sensory evaluation resulted in a determination coecient
(R2 = 0.8443), which showed that 84.43% of the change
could be explained [29]. e lack of t, corresponding to
p-values of 0.0818, showed non-signicance of dierence,
demonstrating that the experimental data was highly
probable. Among the factors explored in the sensory
evaluation of seedless wampee wine, inoculum size (X1)
had the greatest eect followed by initial sugar (X2), fer-
mentation time (X3), and fermentation temperature (X4).
e combined eects of the tested factors on the sensory
scores were visualized in Fig.2. e quadratic term (X4²)
displayed highly signicance (p<0.0001), followed by X22
and X32 (p < 0.01), and X4² was also signicant (p < 0.05).
According to the response surface and the regression
equation, the optimal value for inoculum concentration,
initial sugar, fermentation temperature and fermenta-
tion time to produce a sensory score value of 94.68
were 0.63%, 200.47g/L, 22.00°C and 9.06 d. To ensure
the validity of the model equations, three replicate tests
were performed under the optimal conditions with slight
modication as follows: an inoculum concentration of
0.6%, initial sugar concentration of 200g/L, fermentation
at a temperature of 22°C, and fermentation for 9 d, tak-
ing into account the feasibility of the practical operation.
e sensory score of 94.54 was in line with the expected
results, suggesting that the established prediction model
could eectively predict the sensory score.
Amino acid content of the seedless wampee wine during
fermentation
As one of the precursors of volatile compounds, amino
acids are recognized for their contribution to the aroma
and taste of wine [18, 19]. e sensitivity of amino acids
proles to processing conditions varies depending on
the processing methods and materials [30]. Fifteen free
amino acids were detected in seedless wampee wine in
this study. ese fatty acids were categorized accord-
ing to taste as sweet amino acids (Ser, Ala, r, Gly, Cys,
Pro), bitter amino acids (Leu, Ile, Val, His, Arg, Lys, Tyr),
and umami amino acid (Asp, Glu) [31]. Overall, there
was a greater variation in total amino acids during the
Table 2 Variance analysis of response surface model
Source Sum of
square
De-
gree of
freedom
Mean
square
F-value P-value
Model 175.32 14 12.52 5.42 0.0016*
X19.72 1 9.72 4.21 0.0595
X23.85 1 3.85 1.67 0.2175
X31.33 1 1.33 0.58 0.4600
X40.75 1 0.75 0.32 0.5779
X1 × 27.29 1 7.29 3.16 0.0974
X1 × 30.022 1 0.022 9.74 × 10− 3 0.9228
X1 × 41.32 1 1.32 0.57 0.4618
X2 × 31.21 1 1.21 0.52 0.4812
X2 × 44.84 1 4.84 2.10 0.1698
X3 × 40.30 1 0.30 0.13 0.7229
X1² 15.90 1 15.90 6.88 0.0200*
X2² 21.98 1 21.98 9.51 0.0081*
X3² 34.04 1 34.04 14.73 0.0018*
X4² 125.06 1 125.06 54.13 < 0.0001**
Residual 32.34 14 2.31
Lack of t 29.67 10 2.97 4.44 0.0818
Pure Error 2.67 4 0.67
Cor total 20.74 28
R² 0.8443
*means signicant dierence (p < 0.05), **means extremely signicant
dierence (p < 0.01) .

Page 6 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
main fermentation, ranging from 100.98mg/L on day 5
to 2492.36mg/L on day 0, but the levels remained rela-
tively stable during the post-fermentation period, ranging
from 187.38 to 210.74mg/L (Fig.3a). Regarding amino
acids responsible for taste, the percentage of sweet amino
acids decreased from 82% on day 0 to 48% on day 29,
which could be partly attributed to the signicant reduc-
tions of Ser and Ala (Fig.3b). On the 9th day, the volatile
Fig. 2 3D surface plots for the eect of independent variables (a. inoculum concentration and initial sugar; b. inoculum concentration and fermentation
time; c. inoculum concentration and fermentation temperature; d. initial sugar and fermentation time; e. initial sugar and fermentation temperature; f.
fermentation time and fermentation temperature.) on sensory score of seedless wampee wine

Page 7 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
compounds had 52.74% of sweetness amino acids, 12.12%
of umami amino acids and 35.07% of bitterness amino
acids. Although the proportion of bitter amino acids
increased during the initial stage of fermentation, their
concentration decreased signicantly during fermenta-
tion, dropping from 161.18mg/L on day 0 to 54.93mg/L
on day 29, which represents a 65.9% reduction. It has
been suggested that the bitterness threshold is adjusted
through the acidity threshold [32], and that the increase
of bitter amino acids may balance the acidity of seed-
less wampee, resulting in a more harmonious avor of
wampee wine. Furthermore, the percentage of umami
amino acids signicantly increased during fermentation,
from 11% on day 0 to 22% on day 29. Previous studies
Fig. 3 Changes in amino acid composition (a) and the proportion of taste amino acid (b) during seedless wampee wine fermentation

Page 8 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
have demonstrated that fermented beverages with pro-
longed yeast exposure contain high levels of free Glu,
which may enhance umami more than beverages with
limited or no yeast exposure. is is consistent with the
results of our study, where the freshness amino acids
increased signicantly during the late fermentation of
wampee wine [33]. Amino acids not only contribute to
aroma formation, but are also precursors of a variety of
avor compounds, mainly due to their role in microbial
growth and metabolism as nitrogen sources [17]. e
prole of individual amino acids in fruit wine was inu-
enced by various factors, including yeast, fermentation
conditions, and carbon source [34, 35]. In our study,
decreases in bitter amino acid content during fermen-
tation might contribute to the taste of seedless wampee
wine.
Organic acid content of the seedless wampee wine during
fermentation
e presence of an adequate amount of organic acids has
been shown to hinder the growth of contaminating bac-
teria and improve the mellowness and avor of wine [36].
In order to investigate the variations in organic acid lev-
els in the fermentation process of seedless wampee wine,
eight organic acids, namely oxalic acid, tartaric acid,
pyruvic acid, malic acid, lactic acid, acetic acid, citric acid,
and succinic acid, were identied through HPLC analysis
of the seedless wampee wine. Overall, the concentration
of organic acids exhibited a modest decline during the
fermentation process and then maintained a relatively
stable, uctuating between 15.24 and 16.08 mg/L after
the seventh day, suggesting that the microbial commu-
nity involved in the fermentation of wampee wine had
achieved a state of equilibrium (Fig.4a).
As for the concentrations of individual organic acids,
tartaric acid, lactic acid, and succinic acid, were observed
to be signicantly increased (p < 0.05). Tartaric acid
exhibited the highest increase, rising from 0.14 mg/L
on day 0 to 2.05mg/L on day 29, indicating a 13.64-fold
increase. e peel of seedless wampee is a possible source
of the higher tartaric acid concertation [37]. On the other
hand, the levels of oxalic acid, pyruvic acid, malic acid,
acetic acid, and citric acid were signicantly reduced
(p < 0.05), and malic acid exhibited the most signicant
decline, decreasing from 0.22mg/L on day 0 to 0.04mg/L
on day 29.
After the fermentation process, the predominant
organic acid found in seedless wampee wine was changed
from initially acetic acid to lactic acid, with the percent-
age of acetic acid decreasing from 54% on day 0 to 28% on
day 29 and the percentage of lactic acid increasing from
23% on day 0 to 34% on day 29 (Fig.4b). e increase in
alcohol content during fermentation could lead to the
solubility of lactic acid in wampee. As reported, most
organic acids in beverages were not directly correlated
with sensory characteristics, however, the ratio of ace-
tic acid to total organic acid content exhibited a strong
correlation with sensory characteristics [38]. e ratio of
acetic acid to total organic acid content remained steady
(22-28%) from day 5 to day 29, suggesting that organic
acids have a minimal impact on the sensory features of
seedless wampee wine during this stage. Furthermore, it
should be noted that acetic acid contributes to the syn-
thesis of ethyl acetate, and the reduction of acetic acid
during fermentation is accompanied by an increase in
ethyl acetate concentration, which may ultimately result
in improved fruit aroma of wampee wine [39].
Dynamic changes of aroma compounds in seedless
wampee wine during fermentation
ere was a strong relationship between the sensorial
properties and aroma compounds of fruit wine [40]. e
main volatile aroma compounds of seedless wampee
wine during fermentation were determined by HS-
SPME-GC–MS system, including 14 esters, 10 alcohols,
27 terpenes, and 3 acids (Table3). In general, the com-
position of volatile aroma components varied during the
fermentation process. e avor components of seedless
wampee fruit presented a fruity and oral aroma that is
characteristic of terpenes, with lower levels of alcohols,
esters and acids [41]. During the fermentation process,
more than10 esters, 3 alcohols, 2 acids and 4 terpenes
being produced in seedless wampee wine, whereas 9 ter-
penes found in wampee juice were not detected in the
resulting wine. e fermentation process resulted in the
gradual development of a delicate and mellow avor of
seedless wampee wine, which was achieved by day 29.
Esters play a major role in providing fresh and fruity
fragrances to wine. ey are primarily produced dur-
ing yeast metabolism through the fatty acid acyl- and
acetyl-coenzyme A (CoA) pathways [42, 43]. e seed-
less wampee wine contained twelve esters, eight of which
were ethyl esters of fatty acid. Ethyl decanoate and ethyl
octanoate were promoted most signicantly after fer-
mentation compared with seedless wampee juice, fol-
lowed by ethyl 9-decenoate, ethyl palmitate and ethyl
tetradecanoate (> 1%). e similar trend of change for
ethyl decanoate and ethyl octanoate was observed dur-
ing wine fermentation, which brought out grape and fat
odors to the seedless wampee wine [23].
e alcohols present in fruit wine that are derived
from yeast`s amino acid metabolism are associated
with the variety of fruit [44]. Phenylethyl alcohol and
n-pentanol were the prominent higher alcohols found
in seedless wampee wine as they were the byproducts
of alcoholic fermentation. A moderate amount of these
compounds contributes to the mellow and sweet taste
of fruit wine. For instance, phenylethyl alcohol is known

Page 9 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
for its rose-like aroma and jasmine aroma [45], while
n-pentanol plays a role in providing bitter almond and
fat avor [46]. Additionally, various alcohols including
4-terpinenol, linalool, (-)-α-cadinol, spathulenol, and
α-bisabolol were derived from wampee juice, although
there were some losses during the fermentation process.
Terpenes have a unique aroma with a low avor
threshold and are reported as the characteristic a-
vor for ripened fruit and wine [47]. In seedless wampee
Fig. 4 Changes in organic acid composition (a) and the proportion of organic acid (b) during seedless wampee wine fermentation

Page 10 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
Category Aroma component RI values Published
RI values
Fermentation time (day)
0 2 3 5 7 9 14 19 24 29
Esters L-bornyl acetate 1296 1283 0.42 ± 0.03 1.53 ± 0.06 1.06 ± 0.27 nd nd nd nd 0.67 ± 0.15 0.53 ± 0.03 0.60 ± 0.06
ethyl palmitate 2191 2137 0.06 ± 0.01 nd 0.10 ± 0.04 0.25 ± 0.13 0.19 ± 0.01 1.49 ± 0.32 0.23 ± 0.03 0.39 ± 0.03 1.13 ± 0.28 1.36 ± 0.84
isoamyl acetate 1369 1356 nd 1.18 ± 0.76 0.66 ± 0.15 1.26 ± 0.51 0.65 ± 0.11 1.03 ± 0.05 0.45 ± 0.08 0.35 ± 0.16 0.62 ± 0.02 0.61 ± 0.02
ethyl acetate 1209 1237 nd 0.12 ± 0.02 nd 0.03 ± 0.02 0.17 ± 0.04 0.23 ± 0.03 0.06 ± 0.02 0.07 ± 0.01 0.06 ± 0.04 0.03 ± 0.02
ethyl decanoate 1668 1657 nd nd 6.74 ± 0.46 12.74 ± 2.44 14.97 ± 0.4 0.20 ± 0.01 15.86 ± 1.33 15.83 ± 0.62 15.64 ± 1.46 15.05 ± 0.15
ethyl octanoate 1392 1384 nd nd 3.10 ± 0.85 10.48 ± 0.81 7.25 ± 0.35 0.13 ± 0.01 10.21 ± 0.21 6.15 ± 2.89 8.51 ± 1 8.2 ± 0.04
ethyl laurate 1313 1349 nd nd 1.35 ± 0.56 4.36 ± 0.57 4.28 ± 0.33 0.28 ± 0.05 4.58 ± 0.01 6.74 ± 0.01 7.72 ± 1.11 0.16 ± 0.01
isoamyl decanoate 1628 1579 nd nd nd 0.14 ± 0.01 nd 0.67 ± 0.17 0.14 ± 0.01 nd nd 0.2 ± 0.02
ethyl tetradecanoate 1571 1649 nd nd nd nd 0.5 ± 0.04 0.48 ± 0.14 0.57 ± 0.03 1.31 ± 0.02 2.59 ± 0.69 1.04 ± 0.17
ethyl 9-decenoate 1747 1764 nd nd nd nd nd 5.62 ± 0.28 2.39 ± 1.33 5.09 ± 0.30 3.84 ± 0.38 3.45 ± 0.21
isoamyl octanoate 2108 2186 nd nd nd 0.96 ± 0.40 1.39 ± 0.17 0.96 ± 0.03 1.03 ± 0.29 1.17 ± 0.03 8.51 ± 0.07 0.51 ± 0.10
ethyl nonanoate 1288 1367 nd nd nd nd nd nd nd nd 0.06 ± 0.01 0.08 ± 0.01
ethyl caproate 1357 1425 nd nd 0.92 ± 0.07 1.62 ± 0.48 0.73 ± 0.04 0.25 ± 0.06 0.96 ± 0.18 nd nd nd
propyl caprylate 1349 1394 nd nd nd nd nd 0.24 ± 0.04 0.06 ± 0.01 nd 0.17 ± 0.02 nd
Alcohols 4-terpinenol 1820 1856 9.92 ± 0.26 4.92 ± 0.36 nd 2.71 ± 0.95 3.21 ± 0.37 0.61 ± 0.22 nd nd 0.03 ± 0.19 5.06 ± 0.28
linalool 1537 1438 3.10 ± 0.34 1.56 ± 0.40 1.35 ± 0.32 0.83 ± 0.17 0.6 ± 0.21 5.87 ± 0.48 nd nd 0.67 ± 0.15 1.01 ± 0.15
(-)-α-cadinol 1336 1346 1.42 ± 0.05 0.98 ± 0.38 1.88 ± 0.04 0.61 ± 0.06 0.43 ± 0.26 0.28 ± 0.03 0.36 ± 0.21 0.31 ± 0.05 0.26 ± 0.14 0.96 ± 0.08
spathulenol 1676 1598 0.39 ± 0.02 nd 0.54 ± 0.47 nd nd nd 0.09 ± 0.03 nd nd 0.31 ± 0.01
α-bisabolol 1412 1436 0.22 ± 0.06 0.18 ± 0.03 0.19 ± 0.1 0.13 ± 0.02 0.07 ± 0.02 0.36 ± 0.33 0.08 ± 0.01 0.3 ± 0.11 nd 0.15 ± 0.04
palustrol 1295 1357 nd 0.41 ± 0.15 0.15 ± 0.03 0.45 ± 0.14 0.33 ± 0.02 2.48 ± 0.24 0.33 ± 0.04 0.5 ± 0.01 0.59 ± 0.08 1.19 ± 0.05
phenethyl alcohol 2152 2048 nd nd 0.42 ± 0.12 0.62 ± 0.10 0.99 ± 0.3 0.55 ± 0.02 0.36 ± 0.05 0.44 ± 0.05 0.39 ± 0.07 0.66 ± 0.11
santalol 1612 1698 nd nd nd 0.11 ± 0.03 0.09 ± 0.04 0.44 ± 0.10 nd nd 0.14 ± 0.04 0.10 ± 0.01
n-pentanol 1581 1548 nd nd 0.35 ± 0.14 nd nd nd 1.07 ± 0.48 nd nd 1.51 ± 0.16
α-terpineol 1430 1364 nd nd nd nd 0.04 ± 0.02 2.19 ± 0.14 nd nd nd nd
Terpenes α-ocimene 1921 1953 12.93 ± 0.38 nd nd nd 1.95 ± 0.27 0.27 ± 0.18 nd nd nd 1.98 ± 0.56
α-phellandrene 1744 1648 9.94 ± 0.20 nd nd nd nd nd 3.23 ± 1.09 3.23 ± 0.32 3.54 ± 1.73 2.33 ± 0.04
4-carene 1418 1358 4.99 ± 0.24 nd nd nd nd 1.69 ± 0.17 1.62 ± 0.43 1.40 ± 0.06 0.84 ± 0.38 1.80 ± 0.12
calamenene 1433 1488 3.14 ± 0.16 nd nd nd nd nd 0.25 ± 0.01 0.35 ± 0.07 0.28 ± 0.10 0.44 ± 0.01
α-pinene 1211 1269 2.72 ± 0.37 0.14 ± 0.02 1.78 ± 0.57 0.7 ± 0.06 0.65 ± 0.34 1.29 ± 0.25 0.73 ± 0.28 0.19 ± 0.03 1.41 ± 0.06 1.10 ± 0.24
cadinene 1406 1385 1.40 ± 0.08 nd nd nd nd nd 0.28 ± 0.18 nd nd 1.34 ± 0.78
β-sesquiphellandrene 1586 1648 0.66 ± 0.10 4.17 ± 0.12 2.84 ± 0.2 3.33 ± 0.18 3.28 ± 0.27 nd nd 3.37 ± 0.15 3.41 ± 0.53 2.55 ± 0.25
α-caryophyllene 1725 1665 0.54 ± 0.22 nd nd nd 1.73 ± 0.3 nd 2.43 ± 0.59 2.00 ± 0.75 0.42 ± 0.07 0.95 ± 0.25
terpinolene 1337 1354 0.28 ± 0.10 nd 1.49 ± 0.07 0.15 ± 0.08 0.25 ± 0.08 0.30 ± 0.14 0.34 ± 0.28 0.15 ± 0.02 0.14 ± 0.05 0.14 ± 0.03
α-cedrene 2050 1954 0.10 ± 0.03 nd 0.11 ± 0.05 0.13 ± 0.04 0.11 ± 0.01 0.29 ± 0.09 nd nd 0.13 ± 0.09 0.21 ± 0.04
α-farnesene 2005 2045 0.06 ± 0.04 1.28 ± 0.04 4.08 ± 1.54 0.21 ± 0.13 1.01 ± 0.03 1.37 ± 0.16 0.62 ± 0.52 0.66 ± 0.6 1.39 ± 0.19 0.21 ± 0.12
camphene 2106 2164 0.04 ± 0.02 nd nd nd nd nd nd nd nd 0.22 ± 0.15
α- piperidine 1241 1268 nd nd 0.97 ± 0.49 1.22 ± 0.54 0.65 ± 0.04 0.31 ± 0.10 0.58 ± 0.12 0.8 ± 0.23 0.22 ± 0.02 1.77 ± 0.15
α-sabinene 1737 1795 nd 0.17 ± 0.06 nd nd nd nd nd nd nd 1.21 ± 0.56
Table 3 Relative contents (%) of aroma components in seedless wampee wine during fermentation process

Page 11 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
Category Aroma component RI values Published
RI values
Fermentation time (day)
0 2 3 5 7 9 14 19 24 29
ledenee 1588 1649 nd nd 0.54 ± 0.09 nd nd nd nd 0.64 ± 0.04 0.65 ± 0.09 1.52 ± 0.61
(+)-aromadendrene 1433 1498 nd 0.23 ± 0.11 0.25 ± 0.1 0.15 ± 0.02 nd nd nd nd 0.06 ± 0.03 0.26 ± 0.04
(-)-germacrene 1378 1397 1.22 ± 0.10 nd nd nd nd nd nd nd nd nd
calarene 1639 1578 0.82 ± 0.27 nd 0.81 ± 0.31 0.59 nd nd nd nd 0.11 ± 0.01 nd
guaiene 1168 1248 0.59 ± 0.16 nd nd nd nd nd nd nd nd nd
terpinene 2016 1974 0.56 ± 0.15 nd nd nd nd nd nd nd nd nd
caryophyllene 1350 1387 0.52 ± 0.03 nd nd nd nd 2.28 ± 0.23 2.43 ± 0.59 nd nd nd
cedrene 1409 1354 0.45 ± 0.01 nd nd nd nd nd nd nd nd nd
bergamotene 1730 1784 0.39 ± 0.14 nd nd nd nd nd nd nd nd nd
limonene 1724 1648 0.18 ± 0.08 nd nd nd nd nd nd nd nd nd
styrene 1921 2048 0.15 ± 0.06 nd nd nd nd nd nd nd nd nd
cadinene 2475 2454 nd 8.47 ± 0.59 0.75 ± 0.34 0.51 ± 0.08 0.51 ± 0.08 nd nd nd nd nd
caryophylene oxide 2150 2104 nd 0.08 ± 0.02 0.06 ± 0.01 0.08 ± 0.03 0.10 ± 0.03 nd 0.06 ± 0.01 0.05 ± 0.01 nd nd
Acids decanoic acid 1988 1975 nd 1.57 ± 0.25 1.52 ± 0.60 0.63 ± 0.03 0.6 ± 0.05 0.60 ± 0.07 0.25 ± 0.03 0.23 ± 0.04 0.22 ± 0.04 0.45 ± 0.12
octanoic acid 1433 1348 nd 2.74 ± 0.66 2.28 ± 0.89 1.60 ± 0.37 1.03 ± 0.29 0.40 ± 0.07 0.38 ± 0.04 nd nd 0.4 ± 0.01
lauric acid 1643 1687 nd 0.46 ± 0.04 0.38 ± 0.15 0.19 ± 0.03 0.23 ± 0.05 0.40 ± 0.14 0.08 ± 0.01 0.06 ± 0.01 nd nd
nd means not de tected
Table 3 (continued)

Page 12 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
wine, there was an overall downward trend in terpenes
(from 41.68 to 18.03%) compared to day 0. is decrease
could be attributed to the sharp decline in α-ocimene,
α-phellandrene, 4-carene, calamenene and α-pinene. A
decline in terpenes during wine fermentation was attrib-
uted to either volatility or transformation into dierent
metabolites [48]. Additionally, the release of glycocide-
bound terpenes in fruit by enzymatic hydrolysis during
fermentation may partially explain the accumulation of
terpenes in wampee wine [49].
Multivariate statistical analysis of seedless wampee wine
during fermentation
Multivariate data analysis was carried out to analyze the
avor composition, including volatile aroma components
and non-volatile aroma components (amino acids and
organic acids), to map the samples from seedless wampee
wine fermentation and gain understanding of the basic
principles underlying the dierences observed (Fig.5).
According to the results of the principal component
analysis (PCA), the samples from dierent fermentation
periods were distributed across four quadrants (Fig.5a).
e wampee juice samples were situated in the third
quadrant, while the samples that underwent fermenta-
tion for 2–9 days could be found in the rst and second
quadrants, and the samples that fermented for 14–29
days were situated in the fourth quadrant. ree distinct
regions were observed in the seedless wampee wine:
unfermented, main fermented and post-fermented phase,
indicating signicant variations in the avor compounds,
including organic acids, amino acids and volatile avor
compounds among the dierent fermentation stages.
To further characterize these samples, a partial least
squares - discriminant analysis (PLS-DA) model con-
trasted with an R2 of 98.4% and a Q2 of 93.9% (Fig.5b).
Clearly, the unfermented samples, as well as those fer-
mented for 2–9 days and 14–29 days, exhibited distinct
characteristics, and the avor variations observed in the
dierent stages of wampee fruit wine fermentation were
clearly separated. ese ndings were consistent with the
results obtained from the PCA model. After conducting
the alignment test and 200 alignment experiments, it was
found that the intersection of the Q2 regression line with
the vertical axis was less than 0, and the y-values of the
left simulation points of R2 and Q2 were lower than the
rightmost origin (Fig.5c). ese results indicated that the
Fig. 5 Multivar iate statistical analysis of seedless wampee wine during seedless wampee fermentation. (a) Principle component analysis (PCA) score plot,
(b) Partial least squares - discriminant analysis (PLS-DA) score plot, (c) Model validation diagram and (d) Variable importance plot(VIP)through PLS-DA
analysis

Page 13 of 14Wang et al. Applied Biological Chemistry (2024) 67:81
PLS-DA model had strong predictive ability with no signs
of overtting. erefore, it can used for avor analysis of
seedless wampee wine. e VIP values indicate the vary-
ing contributions of dierent avor compounds, with
a VIP value greater than 1 indicating a more signicant
discriminatory contribution. A total of 33 major avor
compounds, comprising 6 organic acids, 12 amino acids,
and 15 volatile avor compounds, processed a VIP value
above 1 (Fig.5d). Organic acids, including citric acid, suc-
cinic acid and tartaric acid, along with amino acids such
as Ser, Ala and Asp, and volatile avor compounds such
as cedrene, (-)-germacrene and calamenene, comprised
the vital avor components of seedless wampee wine.
e present study investigated the application of seed-
less wampee in fruit wine fermentation. e optimal
fermentation conditions for seedless wampee wine were
established, including an inoculum concentration of
0.6%, an initial sugar level of 200g/L, a fermentation tem-
perature of 22°C, and a fermentation period of 9 days.
Under these conditions, the sensory score can reach
94.68. en the changes of physicochemical prole and
sensory properties of seedless wampee wine were evalu-
ated under optimal fermentation conditions. Notably,
the non-volatile components, including amino acids and
organic acids exhibited signicant changes during the
main-fermented process. Regarding volatile aroma com-
ponents, the number and concentration of esters showed
a signicant increase after fermentation, whereas the
number and content of terpenes relatively decreased in
seedless wampee wine. ese results enhance our under-
standing of the avor formation of seedless wampee
wine. Further studies could focus on the bioactive com-
ponents and potential health benets of seedless wampee
wine.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13765-024-00938-y.
Supplementary Material 1
Author contributions
Hong Wang: Methodology; Writing — original draft; Writing — review &
editing, Visualization. Xiang Liao: Writing — original draft; Writing — review &
editing; Visualization. Chunyao Lin: Methodology; Software; Writing — original
draft; Writing — review & editing. Weidong Bai: Writing — review & editing;
Supervision. Gengsheng Xiao: Funding acquisition; Supervision. Xingyuan
Huang: Investigation. Gongliang Liu: Funding acquisition. All authors reviewed
the manuscript.
Funding
This research was funded by the Research Capacity Enhancement Project
of Key Disciplines in Guangdong Province, grant number 2021ZDJS005,
Guangdong Provincial Postgraduate Education Innovation Program Project,
grant number 2023SFKC_057, and Guangdong Provincial Key Laboratory
of Lingnan Specialty Food Science and Technology, grant number
2021B1212040013.
Declarations
Conict of interest
The authors declare no conict of interest.
Author details
1Guangdong Provincial Key Laboratory of Lingnan Specialty Food
Science and Technology, College of Light Industry and Food Technology,
Zhongkai University of Agriculture and Engineering, Guangzhou
510225, China
2Key Laboratory of Green Processing and Intelligent Manufacturing of
Lingnan Specialty Food, Ministry of Agriculture and Rural Aairs, Academy
of Contemporary Agricultural Engineering Innovations, Zhongkai
University of Agriculture and Engineering, Guangzhou 510225, China
3Guangdong Xingyao Biotechnology Co. Ltd, Yunfu, Guangdong, China
Received: 5 February 2024 / Accepted: 2 September 2024
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