LWT - Food Science and Technology 145 (2021) 111261

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LWT
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Effect of selected yeast on physicochemical and oenological properties of

blueberry wine fermented with citrate-degrading Pichia fermentans
Wu Zhong a, Shaoquan Liu b, Hong Yang a, Erhu Li a, c, *
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
b
Department of Food Science and Technology, National University of Singapore, Science Drive 2, Singapore, 117543, Singapore
c
Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan, 430070, China

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

Keywords: Blueberry wine has an excessively sour taste due to the presence of high-level of organic acids. Pichia fermentans
Blueberry wine JT-1-3 with a strong ability to degrade citric acid was selected for blueberry wine production in this work. The
Deacidification flocculation of JT-1-3 in comparison with two S. cerevisiaes strains was evaluated. Then citric acid degradation of
Fermentation
JT-1-3 was assessed in synthetic media with different citric acid/glucose ratios under shaking or static condi­
Non-saccharomyces
Organic acid
tions. Finally, JT-1-3 was inoculated in blueberry juice for fermentation. The results confirmed that JT-1-3 had
appropriate flocculation and consumed citric acid and glucose greatly. In final wines, 6.25% (v/v) alcohol was
achieved with 1.83% (w/w) residual sugar. Compared with juice, citric, malic and tartaric acids declined
significantly by 43.35%, 35.29% and 44.68% in wine, respectively. 2-phenylethanol, ethyl 9-hexadecenoate,
ethyl decanoate were the main volatiles in blueberry wine and most esters contributed to the aroma with
high odor activity values. Sensory evaluation showed that wine produced by JT-1-3 imparted improved taste
with lower acidity, bitterness and hotness perceptions compared with S. cerevisiae fermentation. This work
revealed that JT-1-3 had a great potential to be applied for blueberry wine fermentation along with
deacidification.

1. Introduction such as inhibiting protein synthesis of human gingival fibroblasts and

Blueberry is normally consumed fresh, but availability is often
restricted due to harvest seasons and short shelf life; thus, blueberry
wine provides an alternative method to preserve nutrition and extend
sales time. Blueberry wine with attractive color and unique sensory
properties is purported to be helpful for improving ulcer or hemorrhoid
conditions and protecting against “cataracts” (Jagtap & Bapat, 2015;
Santos et al., 2016; Yang et al., 2012). However, blueberry wine fer­
mented with industrial S. cerevisiae often has an excessively sour taste,
which is caused by high levels of residual organic acids, and this might
lead to poor flavor and quality. Citric acid is the major organic acid in
blueberries, comprising over 50% of the total acids; it is also the main
acid in blueberry wine (Famiani et al., 2005; Forney, Kalt, Jordan,
Vinqvist-Tymchuk, & Fillmore, 2012). Apart from the endogenous citric
acid, yeasts would also produce this acid during fermentation via the
tricarboxylic acid (TCA) cycle, leading to a high level of citric acid in
final wine. An excessive amount of citric acid will give the wine an
unacceptable tart taste and may even have side effects on human health
causing extracellular acidosis (Lan et al., 1999).
Some non-Saccharomyces, including Pichia genus and Issatchenkia
terricola have been reported to degrade citric acid (Sun, Gong, Liu, & Jin,
2016; Wen, Wang, & Wang, 2011), but few researchers have reported
their application, other than fermentation performance in fruit wine­
making. Recently, non-Saccharomyces are regarded as a positive
contributor to wine flavor by producing medium-chain fatty acids and
ethyl acetate, as well as being a strong producer of 2-phenylethyl acetate
(Comitini et al., 2011; Maturano et al., 2019; Mestre Furlani et al., 2017;
Varela, 2016; Viana, Gil, Genoves, Valles, & Manzanares, 2008). Some
researchers reported that a few non-Saccharomyces even showed stron­
ger tolerance to winemaking conditions compared to S. cerevisiae and
were suitable for fermentation (Padilla, Gil, & Manzanares, 2016;
Zhong, Chen, Yang, & Li, 2020). Various non-Saccharomyces can pro­
duce a range of important enzymes (β-glucosidase, β-xylosidase, prote­
ase and pectinase) to release aroma compounds and the Pichia genus is
observed to possess most of them (Lee, Ong, Yu, Curran, & Liu, 2010);
they also can be utilized to produce low-alcohol wine (Contreras et al.,

* Corresponding author. College of Food Science and Technology, Huazhong Agricultural University, 1 Shizishan Road, Wuhan, Hubei, 430070, China.
E-mail address: erhuli@mail.hzau.edu.cn (E. Li).

https://doi.org/10.1016/j.lwt.2021.111261
Received 6 August 2020; Received in revised form 5 March 2021; Accepted 6 March 2021
Available online 10 March 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
W. Zhong et al. LWT 145 (2021) 111261

2014). allowed to settle for 16 h. The whole sedimentation progress was

One isolate of P. fermentans with a strong ability to degrade citric recorded and photographed.
acid was found and performed well for improving kiwifruit wine quality The flocculation was further evaluated quantitatively (Li, Zhao,
in our previous research (Zhong et al., 2020). This is consistent with Chang, Zhang, & Bai, 2012) as follows: yeast cells grown in the HG
previous reports which indicated that P. fermentans showed good per­ medium were harvested and washed twice with 10 mL of 0.1 M ethylene
formance in low-alcohol grape and orange beverage fermentations and diamine tetraacetic acid (EDTA); the obtained cells were then
increased the formation of higher alcohols and ethyl acetate (Mingor­ re-suspended in 20 mL of de-flocculating buffer (50 mM sodium acetate,
ance-Cazorla et al., 2003). de Melo Pereira et al. (2014) further revealed 0.1 M EDTA, pH 4.5) and the OD600nm was adjusted to around 2.0 by
that P. fermentans could be applied as a good fermentation starter with using a UV spectrophotometer (UV-1750, Shimadzu, Tokyo, Japan) and
novel and desirable flavor profiles. Moreover, the Pichia genus is found recorded as A. Then 20 mL of cell suspension were centrifuged at 2000 ×
to be able to utilize various substrates including some organic acids and g (Avanti JXN-26, Beckman Coulter. Co) and washed twice with
most sugars, even xylose (Agbogbo & Cowardkelly, 2008; Caputo, de-ionized water. Subsequently, the cells were re-suspended in 20 mL of
Quintieri, Baruzzi, Borcakli, & Morea, 2012). P. fermentans has been flocculating buffer (50 mM sodium acetate, 0.1% CaCl2, pH 4.5). After
proved to have an obvious effect on the wine flavor composition both being vortexed for 2 min, 2 mL of re-suspended culture was pipetted into
qualitatively and quantitatively, especially releasing 2-phenylethanol by a 1.0-cm cuvette. The yeast suspension was rested for 20 min and
catabolism of L-phenylalanine (Caputo et al., 2012). OD600nm was measured as B. Flocculation ability was calculated as C=
The aims of the study were to:1) evaluate citric acid metabolism by (1-B/A) × 100%. The experiments were carried out in triplicate for each
P. fermentans JT-1-3 in synthetic media containing different citric acid/ yeast strain.
glucose compositions under shaking/static incubations; 2) investigate
transformation of non-volatile and volatile compounds during blueberry 2.3. Citric acid degradation performance in synthetic media
wine fermentation along with deacidification.
JT-1-3 was inoculated at 106 CFU/mL and incubated into 150 mL of
2. Material and methods WLC, WLG1 and WLG2 media (Table 1) both in shaking and static cul­
tures at 28 ◦ C. The shaking culture was conducted on a shaker incubator
2.1. Yeast strains and culture media at 120 rpm (IS-RSV1, CRYSTAL). Sampling was done at 24 h’s intervals
for 9 days to monitor OD600nm, glucose and citric acid. The experiments
Commercial S. cerevisiae RV002 was provided by Angel Yeast Co. Ltd. were carried out in triplicate.
(Yichang, Hubei, China). P. fermentans JT-1-3 (NCBI ID: MN314412) was
isolated from soil in Zhejiang Citrus Research Institute and deposited in 2.4. Blueberry wine fermentation
China Center for Type Culture Collection (CCTCC) (NO:2018146).
S. cerevisiae NP-7-5 (NCBI ID: MN736547) was isolated from lemon Rabbiteye blueberry (V.ashei Reade) ‘Britewell’ cultivars from
pulps from Ziyang, Sichuan Province, China. Strains RV002, JT-1-3 and Nanjing, Jiangsu Province, China were harvested at commercially
NP-7-5 were isolated on potato dextrose agar (PDA) (Table 1) and pre­ mature stage in August 2018. After harvest, fruits were immediately
cultured in yeast peptone dextrose (YPD) (Table 1) at 28 ◦ C, 120 rpm for transported to our laboratory where they were stored at − 20 ◦ C until
24 h before application. use. Immature and damaged blueberries were discarded and the pulp
was prepared by using a domestic blender (GZ30, Deerma, China).
2.2. Flocculation assay Pectinase (30,000 U/g, Aladdin, Shanghai, China) was added into the
pulp according to the manufacturer’s recommendation at 1 g/kg of
Three milliliters of precultured yeast cells were inoculated into 100 blueberry incubated at 30 ◦ C, 1 h. The final obtained blueberry must was
mL of high glucose (HG) medium (Table 1) at 106 CFU/mL and incu­ pasteurized at 80 ◦ C for 15 min and the sterilization effect was verified
bated at 28 ◦ C, 120 rpm for 48 h. Seven milliliters of culture were by plating the juice on PDA plates. P. fermentans JT-1-3 was inoculated
sampled into a glass tube, mixed for 10 s with a vortex mixer and finally into 150 mL of blueberry must in 250-mL Erlenmeyer flasks at 106 CFU/
mL. Fermentations were carried out at 28 ◦ C for 16 days with a shaking
Table 1 speed of 80 rpm in a shaker incubator until no significant changes were
Compositions of the culture media. monitored in sugars and citric acid within 12 h. Uninoculated blueberry
Medium Compositions (w/v) Purpose Company juice under the same treatment was used as a reference. The final
blueberry wine was stored at − 20 ◦ C until analysis. All fermentations
YPD 1% yeast extract, 2% Yeast pre- Coolaber Co.
yeast peptone, 2% culture Ltd
were carried out independently in triplicate.
glucose
PDA 20% potato, 2% Yeast isolation Qingdao Hope 2.5. Chemical analysis
glucose, 2% agar Bio-Technology
Co. Ltd
2.5.1. Measurement of glucose, total acidity, total sugars, pH and ethanol
High Glucose 5% glucose, 2% yeast Flocculation Aladdin Co. Ltd
(HG) medium peptone, 1% yeast assay content
extract The glucose content was measured by titration with potassium per­
Wallerstein 0.5% yeast peptone, Citric acid Aladdin Co. Ltd manganate (0.1 M) (GB5009.7–2016, Chinese National Standard). The
Laboratory 0.4% yeast extract, 1% degradation total acidity (expressed as g/L of tartaric acid) and total sugars were
Citric acid citric acid, 0.055% performance
(WLC) KH2PO4, 0.0425% KCl,
assessed based on GB/T 15038-2006 (Chinese National Standard). The
medium 0.0125% CaCl2, pH value was measured using a digital pH meter (PB-10, Sartorius,
0.0125% MgSO4, Goettingen, Germany). The ethanol content was measured as described
0.00025% FeCl3, by Zhong et al. (2020). Ethanol analyses were carried out by using an
0.00025% MnSO4
Agilent 6890 gas chromatograph (California, USA) equipped with a
Wallerstein WLC with 1% glucose
Laboratory flame ionization detector. The injector temperature was kept at 280 ◦ C,
Glucose while the detector temperature was maintained 220 ◦ C. Separation was
(WLG1) carried out on a DB-FFAP (Agilent 122–3232, 30.0 m × 250 μm × 0.25
medium μm) capillary column in the constant flow mode. The oven temperature
WLG2 medium WLC with 2% glucose
was programmed as follows: initial 50 ◦ C, from 50 ◦ C (held for 0 min) to

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W. Zhong et al.
100 ◦ C at the rate of 1 ◦ C/min, from 100 ◦ C (held for 2 min) to 240 ◦ C
(held for 2 min) at the rate of 70 ◦ C/min. The injection volume was 5 μL.
2.5.2. Determination of organic acids
The citric, tartaric and malic acids in blueberry juice and wine were
analyzed by using Waters e2695 HPLC equipped with a photodiode
array detector (2998, Waters, Milford, MA, American) (Zhong, Li, Yang,
& Li, 2018). Separation and quantification of organic acids were per­
formed at 215 nm with an Agilent Zorbax SB-Aq C18 column (4.6 mm ×
250 mm, 5 μm particle size). Isocratic elution was performed with 100%
0.05 M potassium dihydrogen phosphate solution and the injection
volume was 5 μL.
2.5.3. Analysis of volatile compounds with GC-MS
Five milliliters of blueberry juice and wine were placed into 10-mL
vials, followed by adding 2.60 g NaCl and 20 μL of cyclohexanone
(0.946 mg/mL, internal standard). The vial was sealed up and equili­
brated in 50 ◦ C water-bath for 10 min, then the volatile compounds were
headspace extracted for 40 min. The volatiles extraction was conducted
by using a manual solid-phase microextraction (SPME) device equipped
with a 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellfonte, PA, USA).
After extraction, the fiber was then thermally desorbed into the injector
port to desorb the analytes at 250 ◦ C for 5 min. GC-MS was carried out in
triplicate as described by Zhong et al. (2020) using an Agilent 7820 gas
chromatograph and quadrupole mass selective detector 5977A (Santa
Clara, CA, USA). The mass spectral ionization temperature was set at
230 ◦ C and the mass spectrometer voltage was 70 eV. The separation
was accomplished on DB-5 column (30 m × 0.25 mm × 0.25 μm). The
temperature program was programmed at 40 ◦ C for 3 min and then
increased to 160 ◦ C (held for 2 min) at the rate of 3 ◦ C per min, and
finally increased to 220 ◦ C (held for 3 min) at 8 ◦ C/min. Volatile com­
ponents were identified in MS libraries (NIST 14) and semi-quantified by
using an internal standard (Gao, Zhang, Regenstein, Yin, & Zhou, 2018).
The OAVs (odor activity values) were used to evaluate the contribution
to the overall aroma for specific volatiles and their values were further
calculated as follows: R = S/T, where S was the measured values of
volatiles and T represented the flavor thresholds of corresponding
volatiles.
2.5.4. Determination of total flavonoids and phenolics
The total flavonoids and phenolics in blueberry juice and wine were
extracted with 70% ethanol (v/v) by using an ultrasound device (KQ-
600DE, Kunshan Ultrasound Instruments Co. LTD). Total flavonoid
contents were determined using the aluminum nitrate colorimetric
method and total phenolic contents were determined using the
Folin–Ciocalteu method (Zhong et al., 2018). The estimation was carried
out in triplicate and the results were averaged.
2.5.5. Analysis of amino acids content
Samples were sent to Societe Generale de Surveillance S.A (SGS)
(Shanghai, China) for amino acid determination by HPLC with fluores­
cence detector (Shimadzu), using a Venusil XSB-C18 column (4.6 mm ×
250 mm, 5 μm, Bonna-Agela, Tianjin, China) (Wei et al., 2018). Before
testing, samples were derivatized (Gómez-Alonso et al., 2007). Essential
amino acids (EAA) analyzed included threonine, valine, methionine,
isoleucine, leucine, phenylalanine, lysine and total amino acids (TAA)
were further summed.
2.5.6. Sensorial analysis
2.5.6.1. Sensory evaluation by a tasting panel. The final obtained blue­
berry wine was assessed (blind) following the procedure reported by
Maturano et al. (2019) in duplicate by a trained panel from the Wine
Sensorial Analysis Department at Huazhong Agricultural University
(Wuhan, China). The panel consisted of 10 individuals (5 males and 5
3
LWT 145 (2021) 111261
females between 23 and 28 years old). For comparison, blueberry wine
produced with RV002 under the same conditions (section 2.4) and
commercial blueberry wine produced by Huazhong Agricultural Uni­
versity were also assessed. Ten milliliters of each wine at room tem­
perature were evaluated and fresh water was used for palate cleansing.
The sensory evaluation test was carried out in a sensory panel room
at 25 ± 2 ◦ C as described by Gao et al. (2020). A total of 14 sensorial
attributes were evaluated: one color descriptor (color intensity), twelve
aroma and taste parameters (mineral, fruity, flowery, aroma intensity,
aroma quality, acidity, sweetness, hotness, bitterness, structure, astrin­
gency, aromatic persistence) and over impression. The intensity of each
attribute was assessed using a non-structured scale from 0 to 5, where
0 indicated that the descriptor was not perceived and values between 1
and 5 indicated the intensity of the descriptors from low to high.
2.5.6.2. Electronic nose analysis. One milliliter of each blueberry wine
was mixed with 49 mL of ultrapure water firstly and then 2 mL of
samples were taken for electronic nose analysis (Gouda, Ma, Sheng, &
Xiang, 2019). The E-nose instrument ALPHA MOS-Fox 4000 (Toulouse,
France) was equipped with 16 metal oxide sensors and functions of the
sensors are listed in STable 1. The samples were injected to the Fox
system with an autosampler Alpha MOS HS100 (Toulouse, France) from
15-mL sealed vials, the acquisition time was 300 s and the flow rate was
150 mL/min. Headspace generation temperature was 40 ◦ C and syringe
temperature was maintained 50 ◦ C. Four replicates for each wine sample
were used for data analysis.
2.6. Statistical analysis
All experimental data were analyzed using GraphPad Prism software
(V.5.0, GraphPad Inc., La Jolla, CA, USA), OriginPro software (2018,
Southampton, MA, USA), and SPSS Statistics software (V.17.0, SPSS
Inc., Chicago, IL, USA). T-test and one-way analysis of variance
(ANOVA) followed by Duncan tests conducted by using SPSS were
performed to determine the significance level at p < 0.05. PCA analysis
was implemented by using SPSS and heat map was performed by using
OriginPro.
3. Result and discussion
3.1. Flocculation assay
P. fermentans JT-1-3 was proved to possess the ability to degrade
citric acid, but its characteristics related to fermentation properties such
as flocculation has been rarely reported. Flocculation is one of the most
industrially important characteristics of brewer’s yeast. The flocculation
process for P. fermentans JT-1-3, S. cerevisiaes RV002 and NP-7-5 is
displayed in Fig. 1. No obvious changes were found among the three
yeasts within 1 h and clear stratification was observed at 3.5 h (Fig. 1).
Sixteen-h was enough to achieve complete flocculation. Flocculent yeast
cells could show a progressive loss of flocculation in nutrient-starved
conditions (Soares, 2011) and the results showed that P. fermentans
JT-1-3 as well as S. cerevisiae could be induced for flocculation. During
this process, the two S. cerevisiaes showed similar flocculation levels, but
JT-1-3 had a significantly lower speed (SFig. 1). This behavior might be
helpful for P. fermentans to avoid sluggish or stuck fermentation caused
by the settlement of yeasts cells before the fermentation was completed
(Govender, Domingo, Bester, Pretorius, & Bauer, 2008).
3.2. Citric acid degradation in synthetic media
P. fermentans JT-1-3 showed a strong deacidification ability when
citric acid was the only carbon source and the presence of glucose may
hinder citric acid degradation (Zhong et al., 2020). As shown in Fig. 2,
JT-1-3 displayed completely different profiles of OD600nm and citric acid
W. Zhong et al. LWT 145 (2021) 111261

Fig. 1. Qualitative flocculation assay for P. fermentans JT-1-3 (J), S. cerevisiae RV002 (R) and S. cerevisiae NP-7-5 (N).

under shaking or static culture conditions, but a similar trend was found
for glucose consumption.
It was found that OD600nm had a dramatic rise when JT-1-3 was
shaking-cultured within 24 h (Fig. 2 A). No obvious differences of
maximum biomass in WLC, WLG1, WLG2 (OD600nm all being above 2.4
after 96 h) were found when JT-1-3 was grown aerobically, being
consistent with previous reports that Pichia produced cell mass
depending on oxygen supply (Agbogbo & Cowardkelly, 2008). However,
the rising OD600nm was observed during the whole static incubation for
JT-1-3 in WLC, WLG1, WLG2 (Fig. 2 B). A larger OD600nm was further
observed in WLG1 and WLG2 than in WLC, which was caused by the
existing glucose in initial media (Fig. 2 B).
The differences in growth curves for JT-1-3 under shaking and static
culture conditions were partly caused by the distinct consumption of
glucose or citric acid and oxygen adequacy. Most sugars were utilized
within 24 h by JT-1-3 under both culture conditions (Fig. 2 C, D). As for
citric acid, a sharp decline was observed in WLC, WLG1 and WLG2 under
shaking culture and most citric acid was degraded within 120 h, being
lower than 1.1 g/L (Fig. 2 E). However, the citric acid degradation
percentage was limited to 58.70%, 13.50%, 17.10% in WLC, WLG1,
WLG2 at 120 h while JT-1-3 was static-cultured with increasing glucose
and the degradation equilibrium was not reached even at 216 h (Fig. 2
F). The results above indicated that JT-1-3 could consume both citric
acid and glucose simultaneously and this made JT-1-3 possible to be
utilized in blueberry wine fermentation for degrading citric acid.
Furthermore, sufficient oxygen would accelerate citric acid metabolism
for JT-1-3.
3.3. Analytical profiles of blueberry juice and wine
3.3.1. Physicochemical parameters
A pure culture of P. fermentans JT-1-3 was inoculated into blueberry
juice for fermentation and deacidification. A slight pH increase was
found in blueberry wine, which was consistent with the significant
reduction of total acids (Table 2). P. fermentans consumed most sugars in
blueberry juice to produce 6.25% alcohol, showing a higher ethanol
yield than previously reported (Agbogbo, Coward-Kelly, Torry-Smith, &
Wenger, 2006). Some selected non-Saccharomyces had been applied to

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W. Zhong et al.
Fig. 2. The dynamic changes of OD600nm (A, B), glucose (C, D) and citric acid (E, F) content in the pure culture of P. fermentans JT-1-3 in WLC, WLG1, WLG2 under
shaking or static culture conditions.
produce low-ethanol wine for decreasing hotness and bitterness per­
ceptions to balance the wine (Ciani et al., 2016). Researchers have
further reported that Pichia showed the most promise in industrial
application for the ability to ferment a wide range of sugars such as
glucose, mannose and xylose (Nigam, 2001). No significant difference
was found between blueberry juice and wine for total flavonoids, but a
significant decline was found in total phenolics (Table 2), because some
specific yeasts could conduct phenolic biodegradation and biosorption
(Chtourou, Ammar, Nasri, & Medhioub, 2004). Our results showed that
5
LWT 145 (2021) 111261
P. fermentans JT-1-3 could conduct alcoholic fermentation, while pre­
serving most flavonoids.
3.3.2. Changes in organic acids
It could be found that three organic acids all declined significantly
after fermentation (Table 3). Citric acid was degraded by 43.35% and
this was also observed in kiwifruit wine fermented by P. fermentans
(Zhong et al., 2020). Furthermore, malic and tartaric acids decreased by
35.29% and 44.68%, respectively. Besides P. fermentans, A. niger koji
W. Zhong et al. LWT 145 (2021) 111261

Table 2 Table 4
Properties of blueberry juice and wine fermented with P. fermentans JT-1-3. Aroma volatile compounds of blueberry juice and wine fermented with
Blueberry juice Blueberry wine
P. fermentans JT-1-3 and detected by gas chromatograph mass spectrometry (GC-
MS).
pH 3.22 ± 0.01b 3.35 ± 0.01a
Total acid 8.82 ± 0.36a 7.05 ± 0.40b PK RT/ Identified volatile compound Blueberry Blueberry
Total sugars (g/100 g) 12.25 ± 0.35a 1.83 ± 0.07b min juice (μg/mL) wine (μg/mL)
Alcohol (%, v/v) n.d. 6.25 ± 1.15 Higher Alcohols
Total flavonoids (mg/mL) 0.19 ± 0.01a 0.19 ± 0.01a 1 0.850 Isobutyl alcohol 18.39 ± 0.17b 22.41 ± 0.50a
Total phenolics (mg/L) 135.33 ± 0.31a 123.66 ± 4.09b 2 1.929 Isoamyl alcohol 103.67 ± 58.71 ± 6.62b
2.09a
n.d.: Not detected. Values are expressed as the mean ± SD of three independent
3 2.766 2-Methyl-1-butanol n.d. 30.31 ± 0.00
experiments. Mean values in the same row with different lowercase letters (a, b)
4 19.338 2-Phenylethanol 246.75 ± 148.76 ±
are significantly different (p < 0.05). 38.53a 31.55b
Subtotal 368.81 260.19

Table 3 Acids
The content and TAVs (taste activity value) of main organic acids in blueberry 5 0.596 Acetic acid 21.19 ± 0.61b 31.59 ± 2.69a
juice and wine fermented with P. fermentans JT-1-3. 6 22.792 Octanoic acid 2.51 ± 0.00 n.d.
7 31.589 n-Decanoic acid 3.03 ± 0.00 n.d.
Organic acids Blueberry Blueberry Organoleptic descriptors 8 51.528 n-Hexadecanoic acid 1.29 ± 0.01a 2.18 ± 1.04a
(g/L) juice wine 9 54.448 6-Octadecenoic acid 1.81 ± 0.08a 3.24 ± 2.58a
Citric acid 1.73 ± 0.08a 0.98 ± 0.02b Mild and refreshing, short 10 54.567 Oleic Acid 3.35 ± 1.02b 11.56 ± 0.89a
aftertaste Subtotal 33.18 48.57
Malic acid 1.53 ± 0.10a 0.99 ± 0.03b Fresh, slightly bitter
Tartaric acid 0.94 ± 0.04a 0.52 ± 0.06b Slightly astringent, strong Esters
sour taste 11 13.683 Ethyl hexanoate 12.51 ± 0.82 n.d.
12 22.928 Ethyl succinate 27.44 ± 3.19 n.d.
Values are expressed as the mean ± SD of three independent experiments. Mean 13 23.617 Ethyl 9-decenoate 13.57 ± 0.70a 10.23 ± 3.06a
values in the same row with different lowercase letters (a, b) are significantly 14 23.762 Ethyl octanoate 29.62 ± 4.11a 8.74 ± 1.33b
different (p < 0.05). 15 25.131 Ethyl decanoate 70.49 ± 4.58a 65.73 ± 1.29a
16 26.401 2-Phenylethyl acetate 20.54 ± 2.42a 5.46 ± 0.15b
17 34.216 Ethyl isopentyl succinate 0.89 ± 0.21a 2.93 ± 0.63a
extract was also found to have a marked impact on decreasing citric and 18 40.927 Ethyl dodecanoate 44.99 ± 6.50a 15.37 ±
tartaric acids (Gao et al., 2021). Organic acids degradation was also 18.39b
greatly affected by fermentation conditions (Lee et al., 2012). The 19 42.848 Isoamyl decanoate 1.68 ± 0.03 n.d.
20 48.161 Ethyl myristate 5.06 ± 0.07a 3.48 ± 1.80a
dominant flavor of organic acids was sourness, followed by bitterness
21 50.367 Ethyl pentadecanoate n.d. 0.99 ± 0.00
and astringency, and their flavor could not be improved too much with 22 50.867 Ethyl 13-methyl- n.d. 2.12 ± 0.00
increased wine pH (Siebert, 1999). Thus, organic acids degradation tetradecanoate
would be an effective way to improve the taste and P. fermentans in this 23 51.750 Ethyl 9-hexadecenoate 179.75 ± 119.14 ±
study behaved well during blueberry wine fermentation. 12.09a 65.68b
24 52.113 Ethyl hexadecanoate 100.78 ± 41.46 ±
5.60a 30.41b
3.3.3. Changes in amino acids profiles 25 54.937 Ethyl linoleate n.d. 17.45 ± 5.21
The amino acids profiles in blueberry juice and wine are listed in 26 55.081 Ethyl 9-octadecenoate 17.27 ± 16.45 ± 9.41a
STable 2. A significant decrease of 79%, 62%, 44%, 25% and 24% was 10.43a
27 55.208 Ethyl oleate 19.75 ± 59.86 ±
found in histidine, leucine, threonine, valine and aspartic acid after
13.99b 14.45a
fermentation, while the others were mostly preserved. The five amino Subtotal 544.34 369.41
acids were also found to be in low contents or even undetectable in
blueberry wine (Ouyang et al., 2017). Glutamic acid was the main amino Terpenes
acid both in blueberry juice and wine, followed by aspartic acid and 28 18.881 (+)-4-Carene 7.29 ± 0.00 n.d.
29 18.899 3-Carene n.d. 6.79 ± 1.85
lysine, and this was also confirmed by Ouyang et al. (2017). The pre­ 30 23.540 D-Sylvestrene n.d. 12.94 ± 0.00
served proline was further found to affect the mouthfeel and balance the 31 23.526 Terpineol 16.83 ± 2.63a 16.89 ± 0.40a
redox of wine (Gómez-Alonso et al., 2007). The results showed that 32 25.221 Citronellol n.d. 2.58 ± 0.23
P. fermentans was efficient in utilizing histidine, leucine, threonine, 33 49.054 β-Farnesene I 1.47 ± 0.00 n.d.
Subtotal 25.59 39.20
valine and aspartic acid while mostly preserving the other amino acids.
Ketones
3.3.4. Changes in aroma compounds 34 32.612 (Z)- 6,10-dimethyl-5,9- n.d. 2.19 ± 0.00
The changes in volatile profiles of blueberry juice and wine are listed undecadien-2-one
in Table 4. A total of 33 compounds were identified in blueberry juice, Subtotal n.d. 2.19

while 38 volatiles were found in blueberry wine. As shown in Table 4, it

Aldehydes
could be found that acids, terpenes and ketones increased while higher 35 16.223 2-Phenylacetaldehyde n.d. 2.26 ± 0.00
alcohols, esters, aldehydes and volatile phenols decreased after 36 24.268 Decanal n.d. 1.47 ± 0.00
fermentation by P. fermentans JT-1-3. Eleven key odorants were quan­ 37 24.379 2,4-Dimethylbenzaldehyde 11.55 ± 7.00 n.d.
38 24.429 3,4-Dimethylbenzaldehyde 19.64 ± 1.30a 14.39 ± 0.96b
tified in OAVs and the data are shown in Table 5.
39 26.849 2-Decenal 4.98 ± 0.38a 4.36 ± 0.43a
40 31.422 2-Undecenal n.d. 5.52 ± 0.00
3.3.4.1. Higher alcohols. Higher alcohols, mostly contributing to the 41 32.425 2-Dodecenal 6.08 ± 1.73 n.d.
fruity aroma, were one of the main abundant volatiles groups both in 42 50.474 (Z)-9-Tetradecenal n.d. 0.07 ± 0.00
Subtotal 42.25 28.07
blueberry juice and wine, accounting for 31.66% and 31.85% of the total
volatiles (Table 4). Only 4 higher alcohols were identified in this study, Volatile phenols
while Yuan et al. (2018) identified over 12 compounds in blueberry 43 22.039 4-Ethyl-henol 12.81 ± 1.55a 6.57 ± 0.62b
wine and found that their production was highly dependent on yeast (continued on next page)

6
W. Zhong et al. LWT 145 (2021) 111261

Table 4 (continued ) flavor balance of wine by introducing into too much vinegary odor in
PK RT/ Identified volatile compound Blueberry Blueberry excess over 167.4 μg/mL (Barata et al., 2011). Acetic acid produced by
min juice (μg/mL) wine (μg/mL) P. fermentans would not be expected to bring much undesirable odor to
44 27.230 4-Ethyl-2-methoxy-phenol 134.89 ± 55.35 ± 7.21b
the wine in this study.
20.35a
45 35.060 3,5-Di-tert-butylphenol n.d. 1.86 ± 0.00 3.3.4.3. Esters. Volatile esters including acetates and ethyl esters,
46 37.354 2,5-Di-tert-butylphenol n.d. 3.02 ± 0.00 although being present in low concentrations, were important to fully
47 37.465 2,4-Di-tert-butylphenol 3.08 ± 0.39a 2.46 ± 1.06a
Subtotal 150.78 69.26
decipher the blueberry aroma, especially for the “sweet” and “fruity”
Total 1164.95 816.89 fragrances (Yuan et al., 2018). Esters formed the most abundant group of
volatiles in blueberry juice and wine, representing for 46.73% and
n.d.: Not detected. Values are expressed as the mean ± SD. Mean values in the
45.22%, respectively (Table 4). Ma, Yan, Wang, Zhang, and Tao (2017)
same row with different lowercase letters (a, b) are significantly different (p <
0.05).
reported that acetate and ethyl esters for medium-chain fatty acids
increased with increasing P. fermentans inoculation ratio. However, the
amounts of esters indigenous to blueberry decreased together with
Table 5 various acetate esters and fatty acid esters during fermentation. This was
The odor activity values (OAV) of major volatile compounds in blueberry juice also converse in kiwifruit wine fermented by P. fermentans (Zhong et al.,
and wine fermented with P. fermentans JT-1-3. 2020). This might be because of different fermentation conditions,
Volatile compounds Odor thresholds Blueberry wine Organoleptic which affected greatly the ester production by P. fermentans (Ma et al.,
(μg/mL) OAV descriptors 2017). An increased formation was only found in five esters and
Alcohols P. fermentans tended to produce ethyl -pentadecanoate, ethyl
Isobutyl alcohol 40 (Chen et al., 0.56 Fruity, wine-like -13-methyl-tetradecanoate and ethyl linoleate. Although being
2014) decreased, ethyl decanoate still had the highest OAV among the six es­
Isoamyl alcohol 300 (Chen et al., 0.19 Banana, fruity, ters in Table 5 and positively affected the aroma in this work. The
2014) sweet
Phenylethyl 10 (Guth, 1997) 14.88 Floral, sweet, rosy
decline for ethyl decanoate was also found in the wine fermented by
alcohol P. kluyveri, but the OAV was limited to 0.45 (Lu, Huang, Lee, & Liu,
2016). These results suggested that P. fermentans JT-1-3 exhibited great
Acids potential in controlling and balancing esters in the whole wine aroma
Acetic acid 167.4 (Barata 0.19 Vinegar, pungent
profile.
et al., 2011)

Esters 3.3.4.4. Terpenes. To date, terpenes are considered more important

Ethyl 9-decenoate 0.2 (Lu et al., 51.15 Fruity than esters to decipher the blueberry aroma (Yuan et al., 2018) and their
2016)
concentrations varied closely with cultivar (Du & Rouseff, 2014). The
Ethyl octanoate 0.25 (Lu et al., 34.96 Fruity, waxy
2016) indigenous volatile terpenes were all degraded to undetectable levels
Ethyl decanoate 0.2 (Lu et al., 328.65 Sweet, waxy, fruity except for terpineol and P. fermentans JT-1-3 produced three new ter­
2016) penes during fermentation (Table 4). Terpineol along with citronellol, as
2-Phenylethyl 0.25 (Guth, 1997) 21.84 Honey, floralrosy important aroma compounds in blueberries (Yuan et al., 2018), both
acetate
increased after fermentation in this work. Terpineol can be released by
Ethyl dodecanoate 1.154 (Lu et al., 13.32 Sweet, waxy, soapy
2016) non-Saccharomyces (Maturano et al., 2015; Sadoudi et al., 2012) and its
Ethyl 2 (Lu et al., 2016) 20.73 Waxy, fruity, increasing content with a relatively high OAV may provide wine with
hexadecanoate creamy pleasant sweet odor (Table 5).
Terpenes
Terpineol 0.25 (Guth, 1997) 67.56 sweet and 3.3.4.5. Aldehydes and volatile phenols. P. fermentans produced four al­
mushroom dehydes in low concentrations and degraded 2,4-dimethylbenzaldehyde
and 2-dodecenal to undetectable levels. The decrease in aldehydes might
be caused by the reduction to respective alcohols and final conversion to
strain, fermentation temperature and oxygen availability. 2-phenyletha­
esters (Sumby, Grbin, & Jiranek, 2010). As for volatile phenols, 4-eth­
nol was the major compound both in blueberry juice and wine. Although
yl-2-methoxy-phenol was the dominant phenol, accounting for over
its content decreased after fermentation, 2-phenylethanol still had a
80% of the total phenols. Aldehydes and volatile phenols were consid­
high OAV in wine (Table 5). A contrary result was reported by Zhuang
ered as undesirable compounds and the results indicated that
(2015) that P. fermentans could increase 2-phenylethanol production,
P. fermentans could improve the wine aroma by degrading them.
which was characterized as a delicate aroma of rose petals. A greater
production was found for isobutyl alcohol and isoamyl alcohol in wine
fermented by S. cerevisiae (Chen, Chia, & Liu, 2014), while only isobutyl 3.4. Sensorial analysis
alcohol increased and the two alcohols had low OAV in this work.
Higher alcohols being over 350 mg/L were considered to bring unde­ Principal component analysis (PCA) was performed in order to
sirable aroma to the wine (Hazelwood, Daran, van Maris, Pronk, & characterize the sensorial attributes of the wines. The wine tasters
Dickinson, 2008; Li, Zhao, et al., 2012). In this study, the higher alcohols analyzed 14 sensorial attributes (Fig. 3). The first component (PC1)
decreased from 368.81 μg/mL to 260.19 μg/mL, possibly contributing to explained 83.73% of the total variability, while the second component
wine aroma. The results revealed that P. fermentans JT-1-3 would bal­ (PC2) explained 16.27%. The attributes that best described PC1 were
ance higher alcohols to a more acceptable level. astringency, bitterness, hotness, aroma quality and overall impression
and these attributes allowed a clear separation of B and C from A. It
3.3.4.2. Acids. Most acids increased after fermentation, except for should be pointed out that S. cerevisiae or P. fermentans fermentation also
octanoic and decanoic acid (Table 4). Wang et al. (2017) and Ma et al. could be separated by PC2, which was described by acidity and other
(2017) both reported that P. fermentans would increase volatile fatty attributes. Compared with S. cerevisiae fermentation, wine produced
acids content. Acetic acid was the main acid identified in blueberry juice with P. fermentans appeared to be defined by sweetness and overall
and wine with a relatively low OAV (Table 5), potentially perturbing the impression. The results indicated that P. fermentans could decrease the

7
W. Zhong et al.
Fig. 3. Principal component analysis (PCA) of sensorial attributes evaluated by
(1) wine tasters and (2) E-Nose in A: fermented by P. fermentans; B: fermented
by S. cerevisiae; C: commercial blueberry wine.
acidity, astringency, bitterness and hotness perceptions to improve the
overall wine sensory.
The differences in the volatiles fingerprint for blueberry wines were
further verified by the electronic nose. E-nose PCA among all studied
groups is presented in Fig. 3, in which, PC1 and PC2 accounted for
83.17% and 16.83% of the total variance. Also, from the loading plot
among the 16 sensors in SFig. 2, it indicated that T70/2 and P30/2
which were responsible for aromatic and organic compounds had the
highest positive loadings on PC1 and PC2. It revealed that wine fer­
mented by P. fermentans plotted in the fourth quadrant with positive PC1
and negative PC2 would produce more aromatic compounds, less
organic compounds such as ethanol compared with S. cerevisiae
fermentation.
4. Conclusion
P. fermentans JT-1-3 could conduct significant citric acid degradation
and produce blueberry wine with 6.25% ethanol. It was efficient in
histidine, leucine, threonine, valine and aspartic acid utilization and
might carry out phenol biodegradation. In addition, JT-1-3 could pro­
duce more volatile acids, terpenes, ketones and balance the higher al­
cohols and esters to a more acceptable level. Blueberry wine produced
with JT-1-3 also had an improved taste compared with S. cerevisiae
fermentation. In conclusion, the current study indicated P. fermentans
JT-1-3 showed great potential to be applied in blueberry wine produc­
tion or fruit wines with high levels of citric acid. Further work may focus
on finding some potential key genes regulating citric acid metabolism.
8
LWT 145 (2021) 111261
CRediT authorship contribution statement
Wu Zhong: Conceptualization, Methodology, Formal analysis,
Investigation, Data curation, Writing – original draft. Shaoquan Liu:
Data curation, Writing – review & editing, Supervision. Hong Yang:
Validation, Resources, Visualization. Erhu Li: Validation, Resources,
Writing – review & editing, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
This research was supported by Fundamental Research Funds for the
National Natural Science Foundation of China (31771947).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2021.111261.
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