LWT - Food Science and Technology 132 (2020) 109929

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LWT
journal homepage: www.elsevier.com/locate/lwt

Effects of pectinase treatment on the physicochemical and oenological

properties of red dragon fruit wine fermented with Torulaspora delbrueckii
Xiaohui Jiang a, Yuyun Lu a, **, Shao Quan Liu a, b, *
a
Department of Food Science and Technology, National University of Singapore, Science Drive 2, 117543, Singapore
b
National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu, 215123, China

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

Keywords: Red dragon fruit is a popular tropical fruit that has been highly prized for its health benefits partially attributed
Red dragon fruit to the high antioxidant content. Nevertheless, besides being consumed fresh, further processing into juice or
Alcoholic fermentation other products is scarce due to its high pectin content that presents a challenge in industrial processing. In this
Torulaspora delbrueckii
study, we evaluated the effects of pectinase pre-treatment on red dragon fruit wine fermented with Torulaspora
Pectinase treatment
delbrueckii. Pectinase enzyme (Pectinex® Ultra SP-L, added at 0.1% v/v) was applied after pasteurization of juice
followed by fermentation with T. delbrueckii at 20 � C for 14 days. Pectinase pre-treatment did not affect the yeast
growth, nor the production of ethanol (8% v/v) and glycerol (6 g/L), but significantly increased juice/wine yield
by 16% v/v. In addition, pectinase treated samples after fermentation remained significantly higher levels of
residual nitrogen containing compounds, indicating unfavorable fermentation conditions and impaired meta­
bolism of the yeast. Moreover, fermented pectinase treated samples possessed the aroma compound profiles with
enriched esters and terpenes but decreased higher alcohols. Furthermore, pectinase treatment increased the total
phenolic content, but decreased betacyanins content and color intensity compared to control sample. The
findings would have practical significance for diversifying products from red dragon fruit.

1. Introduction
Dragon fruit (also known as pitaya or pitahaya) is widely cultivated
in many tropical and sub-tropical regions of the world and is available
all year round in many Southeast Asian countries (Bellec, Vaillant, &
Imbert, 2006). Red dragon fruit is known for its high antioxidant ca­
pacity which can be attributed mainly to the purple red pigment, beta­
lains, found in the skin and pulp of the fruit (Esquivel, Stintzing, & Carle,
2007; Wu et al., 2006). Like most tropical fruits, dragon fruit is highly
perishable. Fermentation into alcoholic beverages or wine could be an
economical way for postharvest preservation and value-add.
Dragon fruits contain high levels of pectic substances in the peel and
pulp (38–47% dry weight) (Liaotrakoon et al., 2013). Application of
pectolytic enzymes that break down pectic substances in cell walls could
improve the extraction and clarification of pulp. In fruit wine produc­
tion, addition of pectolytic enzymes causes important alterations on the
chemical composition of juice that can affect the aroma, color and
antioxidant capacity in fruit wine (Guo et al., 2018, Ma et al., 2018;
Samoticha, Wojdyło, Chmielewska, Politowicz, & Szumny, 2017).
Together with the application of other cell wall degrading enzymes and
selection of yeast strains, it is possible to further diversify the type and
sensory characteristics of alcoholic beverages from different sources to
cater to the ever-evolving consumer market.
To date, publications on the fermentation of red dragon fruit into
alcoholic beverage or treatment with pectinase enzymes are scarce
(Aliaa, Mazlina, & Taip, 2011; Ngyuen, 2014; Truong & Dang, 2016). In
our previous study (Jiang, Lu, & Liu, 2020), Torulaspora delbrueckii
monoculture has been applied successfully to produce red dragon fruit
wine. T. delbrueckii is a popular non-Saccharomyces yeast that is char­
acterized by its low production of acetaldehyde and acetic acid, plus
higher tolerance to osmotic shock (Bely, Stoeckle, Masneuf-Pomarede, &
Dubourdieu, 2008). In addition, T. delbrueckii is a relatively efficient
fermenter and it has also been successfully used to complete mono­
cultural fermentation of low alcohol beverages, such as beer or sparkling
wines (Basso, Alcarde, & Portugal, 2016; Medina-Trujillo et al., 2017).
Compared to fermentation with Saccharomyces cerevisiae and Lachancea
thermotolerans, T. delbruckii fermented red dragon fruit wine contained
significantly lower level of glycerol and acetic acid, higher level of

* Corresponding author. Department of Food Science and Technology, National University of Singapore, Science Drive 2, Singapore.
** Corresponding author.
E-mail addresses: fstluy@nus.edu.sg (Y. Lu), fstlsq@nus.edu.sg (S.Q. Liu).

https://doi.org/10.1016/j.lwt.2020.109929
Received 28 May 2020; Received in revised form 19 July 2020; Accepted 21 July 2020
Available online 22 July 2020
0023-6438/© 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Jiang et al.
Fig. 1. Changes in A) viable yeast cell count, B) � Brix, C) fructose, D) glucose and E) ethanol during fermentation of red dragon fruit juice by T. delbrueckii Biodiva.
Pectinex treatment (▴), control without pectinase treatment (■). a, b Statistical analysis (n ¼ 3) at 95% confidence level with the same letter indicating no sig­
nificant difference.
higher alcohols, isoamyl acetate and terpenes as well as better color
retention (Jiang et al., 2020). The aim of the present study was to
investigate the effect of pectinase pre-treatment on yeast fermentation
performance, physicochemical and oenological properties of red dragon
fruit wine fermented with T. delbrueckii.
2. Materials and methods
2.1. Raw materials preparation
Red dragon fruits (Hylocereus costaricensis) imported from Malaysia
were purchased from a local wholesale market in Singapore. The skins
were removed, and the pulp was prepared using a domestic blender and
stored at 20 � C before use. Torulaspora delbrueckii Biodiva was pur­
chased from Lallemand Inc., Brooklyn Park, Australia. The freeze-dried
yeast cells were propagated in YM broth (containing 2% glucose, 0.25%
yeast extract, 0.25% bacteriological peptone and 0.25% malt extract). at
2
LWT 132 (2020) 109929
25 � C for 48 h and then stored at 80 � C until used. Pectinex® Ultra SP-L
obtained from Novozyme, Demark, contains mainly polygalacturonase,
pectin trans-eliminase and pectin esterase.
2.2. Preparation for pectinase treatment and fermentation
Dragon fruit juice (pH 4.7, � Brix 13.1) was adjusted to pH 4.0 and
�
Brix 20 with 1 M DL-malic acid and sucrose, respectively. The adjusted
juice was then pasteurized at 60 � C for 20 min. Effectiveness of the
pasteurization process was verified by plating on potato dextrose agar
(PDA, Oxoid, Hampshire, England) for yeast checking and de Man,
Rogosa and Sharpe (MRS) agar for bacteria checking. No visible cell
count was found on the PDA and MRS plates. A starter culture was
prepared by inoculating 10% (v/v) yeast pure culture (in YM broth) into
the pasteurized red dragon fruit juice and incubating at 25 � C for 96 h to
achieve a yeast cell count of about 107 CFU/mL. Pectinase treatment
(PT) samples were subjected to 0.1% v/v Pectinex treatment at 40 � C for
X. Jiang et al. LWT 132 (2020) 109929

Table 1 evaporative light scattering detector (ELSD). A Supelcogel C-610 col­

Oenological parameters of dragon fruit wines fermented with T. delbrueckii umn (300 � 7.8 mm, Supelco, Sigma-Aldrich, Barcelona, Spain) was
without (Control) and with Pectinex pre-treatment (Pectinex). used to separate organic acids, glycerol and ethanol. Free amino acids
Day 0 Day 14 were analyzed using an amino acid analyzer (ARACUS, MembraPure,
Control Pectinex Control Pectinex
Hennigsdorf, Germany). The separation was carried out in lithium sys­
tem column kit consisting of a pre-column and a separation column
pH 4.03 � 4.01 � 4.11 � 4.11 �
(150 � 4 mm) using the standard solutions and separation method (for
0.03a 0.03a 0.01b 0.01b
�
Brix 20.11 � 20.40 � 8.25 � 8.70 � physiological amino acids) provided by the manufacturer.
0.01a 0.02b 0.02c 0.12d
Ethanol (% v/v) LOQ LOQ 8.33 � 8.02 � 2.5. Analysis of volatile compounds with GC-MS/FID
0.31a 0.68a
Glycerol (g/L) LOQ LOQ 6.07 � 6.04 �
0.59a 0.29a Volatile compounds were analyzed using headspace solid phase
Sugars (g/L) microextraction (SPME) coupled with gas chromatography-mass spec­
Fructose 67.79 � 72.37 � LOQ LOQ trometer (Agilent 7890A, Santa Clara, CA, USA) and flame ionization
1.24a 3.11b
detector (GC-MS/FID) as reported by Lu, Huang, Lee, and Liu (2015).
Glucose 103.98 � 121.18 � LOQ LOQ
7.97a 6.09b Volatile samples were extracted using a Carboxen/PDMS fiber (85 μm,
Organic acids (g/L) Supelco, Sigma Aldrich, Barcelona, Spain) at 60 � C for 50 min with 250
Acetic acid LOQ LOQ 0.148 � 0.234 � rpm agitation. Separation of volatiles was carried out in an Agilent
0.014a 0.010b DB-FFAP column (60 m � 0.25 mm ID). Volatiles were identified by
Citric acid 0.536 � 0.551 � 0.623 � 0.587 �
0.010a 0.015b 0.033c 0.013c
comparing their MS value with Wiley MS and NIST libraries and verified
Lactic acid 0.827 � 0.964 � 0.751 � 0.755 � by linear retention index (LRI) values. LRI values were determined using
0.071a 0.082a 0.018b 0.088b a series of alkanes (C5–C40) on the DB-FFAP column under identical
Malic acid 11.72 � 11.76 � 6.77 � 6.14 � conditions.
0.80a 0.36a 0.39b 0.024c
Pyruvic acid LOQ LOQ 0.475 � 0.594 �
0.015a 0.033b 2.6. Determination of betacyanin content (BC)
Succinic acid LOQ LOQ 1.269 � 1.202 �
0.049a 0.048a
BC was determined using a spectrometric method as described by
Galacturonic acid LOQ 0.304 � LOQ 0.511 �
0.034a 0.011b
Stintzing, Schieber, and Carle (2003). The absorbance of samples was
Juice/Wine yield (% 48.5 � 2.8a 52.1 � 3.5a 59.2 � 1.0b 75.3 � 1.4c measured at 538 nm using a UV spectrometer (Shimadzu UV mini-1240,
v/v) Kyoto, Japan). The BC, expressed as betanin equivalent (BE), was
Viscosity (cP) 9.39 � 9.40 � 8.04 � 6.53 � 0.03c calculated by the following equation:
0.04a 0.09a 0.06b
A538 � MW � DF � 1000
Pectinex, pectic enzyme. Betacyanin ðmg=LÞ ¼
LOQ: limit of quantification.
ε�L
Values are given as the mean � standard deviation (n ¼ 3) and the different
where A538 is the absorbance at 538 nm; DF is the dilution factor; L is the
letters within each row are significantly different (p < 0.05).
path length of cuvette (L ¼ 1 cm); MW is the molecular weight of betanin
(MW ¼ 550 g/mol); ϵ is the molar extinction coefficient of betanin (ϵ ¼
1 h after the pasteurization and then cooled to 25 � C. For fermentation, 60,000 L/mol*cm).
1% (v/v) starter culture was inoculated into the pasteurized or PT red
dragon fruit juice with the initial yeast cell count of ~105 CFU/mL.
2.7. Total phenolic content (TPC) and total antioxidant capacity (TAC)
Triplicate fermentations of 200 mL juice were carried out at 20 � C for 14
days with sampling at 2 days’ interval. Samples were centrifuged at
Total phenolic contents were determined using a Folin-Ciocalteu
10,000 � g, 10 � C for 10 min (Eppendorf 5810R, Hamburg, Germany)
method as described by Isabelile, Lee, Ong, Liu, and Huang (2008)
before further analysis except for viable yeast cell counts where centri­
with a microplate reader (Multiskan Spectrum, Thermo Scientific, Mil­
fugation was not performed.
ford, MA, USA). The concentration of total phenolic compounds was
expressed as mg/L gallic acid equivalent (GAE) in fresh samples. Total
2.3. Measurement of viable yeast cell count, total soluble solids, viscosity antioxidant capacity was measured by using the oxygen radical absor­
and color bance capacity (ORAC) assay according to Huang, Ou,
Hampsch-Woodill, Flanagan, and Prior (2002) and expressed as mM
The viable yeast cell count was determined using spread plating trolox equivalent (TE).
method on PDA. Total soluble solids (TSS) expressed as � Brix were
measured with a refractometer (ATAGO, Tokyo, Japan). Viscosity of 2.8. Statistical analysis
samples was measured using Brookfield DVII þ viscometer (Middleboro,
MA, USA) with Spindle 61 at 100 rpm. The results were expressed as Statistical analysis was carried out by one-way ANOVA (analysis of
centipoise (cP), which equals to 0.001 Pa*s. Color aspects expressed as variance) and Tukey’s test at 95% confidence level using SPSS 17.0
L*, a* and b* values in CIELab color space were measured by using a software for Windows (SPSS Inc., Chicago, IL, USA). Results were
spectrophotometer (Konica Minolta CM-5, Osaka, Japan) with the D65 considered statistically significant if p < 0.05.
illuminant.
3. Results and discussion
2.4. Analysis of non-volatile components and ethanol with HPLC
3.1. Effect of pectinase pre-treatment on yeast growth and oenological
Samples were filtered through 0.20 μm membrane before HPLC properties
analysis as described in detail in our previous work (Jiang et al., 2020).
Sugars were separated with a Zorbax carbohydrate column (150 � 4.6 The pectinase pre-treatment dosage (0.1% v/v), temperature (40 � C)
mm, Agilent, Santa Clara, CA, USA) in a Shimadzu UFLC system with an and duration (1 h) was determined based on previous studies using

3
X. Jiang et al.
Fig. 2. Changes in A) D-galacturonic acid, B) betacyanin content, C) total phenolic content and D) total antioxidant capacity in red dragon fruit wine during
fermentation by T. delbrueckii Biodiva. Pectinex treatment (▴), control without pectinase treatment (■). a, b Statistical analysis (n ¼ 3) at 95% confidence level with
the same letter indicating no significant difference.
Pectinex® Ultra SP-L to improve the processing of dragon fruit juice
(Aliaa et al., 2011; Truong & Dang, 2016), as well as preliminary eval­
uation of the enzyme activity and reaction kinetics. Similar evolution
patterns in terms of yeast cell counts (Fig. 1A), � Brix (Fig. 1B), sugars
(Fig. 1C and D) and ethanol (Fig. 1E) were observed during red dragon
fruit juice alcoholic fermentation (AF) with and without pectinase
treatment. The final yeast cell counts in PT samples reached the same
level as that of control samples despite slightly lower initial cell count.
Sugars (glucose and fructose) in both PT and control samples were
totally consumed at day 8 or day 10 of fermentation, indicating com­
plete AF by T. delbrueckii. In addition, both treatment and control
samples showed similar final � Brix (8.3–8.7), ethanol level (8.0–8.3%,
v/v) and pH (4.11) values (Table 1). Furthermore, the production of
glycerol, a major by-product of AF, in both samples (~6 g/L) fell into the
normal range of grape wine (4–9 g/L) (Zhao, Procopio, & Becker, 2015).
Pectinase pre-treatment did not cause significant alteration on either the
rate of AF or basic oenological properties, which is in accordance with
the result from several other studies (Alpeza, Ganic, Vanzo, & Herjavec,
2017; Guo et al., 2018; Ma et al., 2018).
It is interesting to note that sucrose added into the juice before
fermentation for the purpose of adjusting � Brix was not detected in any
of the samples at day 0. This was likely due to the activity of endogenous
sucrose invertase present in dragon fruit (Jamilah, Shu, Kharidah,
Dzulkily, & Noranizan, 2011), which rapidly hydrolyzed sucrose into
glucose and fructose.
The quantity and composition of organic acids have great implica­
tions for both the taste and flavor of samples. Addition of malic acid for
4
LWT 132 (2020) 109929
the purpose of adjusting juice pH before AF accounted for the high level
of malic acid in raw juice. At the end of fermentation, malic acid con­
centration dropped to about half of the initial amount (from 11.7 g/L to
6.1–6.8 g/L) (Table 1). T. delbrueckii has been reported to consume
20–25% malic acid (Chen et al., 2018) and the rest of malic acid likely
underwent a passive diffusion process into yeast cells rather than being
metabolized (Coloretti, Zambonelli, Castellari, Tini, & Rainieri, 2002).
Besides malic acid, citric acid and lactic acid were the major organic
acids found in raw red dragon fruit juice (Table 1). No significant dif­
ferences were observed between control and PT samples in the con­
centration of citric, lactic, and succinic acids after AF. Although PT
samples contained significantly higher levels of acetic acid (0.23 g/L)
compared to control sample (0.15 g/L) (Table 1), both are considered
low for grape wine (normally range, 0.2–0.6 g/L). Acetic acid is pro­
duced from oxidation of acetaldehyde by aldehyde dehydrogenase or
hydrolysis of acetyl CoA derived from pyruvate. Yeast cells accumulate
acetic acid under certain adverse environmental conditions (Blomberg &
Adler, 1992). The clarification effect of pectinase may cause the increase
of osmotic pressure in media, which could cause T. delbrueckii to respond
with increased production of acetic acid in order to counterbalance the
osmotic pressure. Additional accumulation of acetic acid could also
come from acetyl residues present in dragon fruit pectin which has been
released due to hydrolysis by pectin acetylesterase activity in Pectinex.
D-Galacturonic acid was only detected in PT samples (Table 1).
Pectinase hydrolyzed pectin molecule through de-polymerization (hy­
drolases and lyases) and de-esterification (esterase) reactions. Gal­
acturonic acid oligomers or monomers are the main products from the
X. Jiang et al. LWT 132 (2020) 109929

Table 2 sample. Pectinase has been used extensively in fruit juice extraction and
Concentration (mg/L) of nitrogen containing compounds in red dragon fruit clarification. It plays important roles in reducing viscosity and
juice and wine fermented with T. delbrueckii without (Control) and with Pectinex increasing yield through liquefaction of pulp and removal of peel.
pre-treatment (Pectinex). Depending on the type of fruit and application conditions, the increase
Nitrogen containing Day 0 Day 14 in final yields varies from 5% to more than 100% (Garg et al., 2016).
compounds
Control Pectinex
3.3. Effect of pectinase treatment on nitrogen containing compounds
Alanine 19.36 � 0.10a 6.55 � 0.43b 16.90 � 2.56c
Ammonium 25.01 � 0.65a 7.50 � 0.69b 14.53 � 1.17c
Arginine 109.03 � 4.95 � 0.23b 37.79 � 2.59c Nitrogen is an essential nutrient for yeast growth and often the rate
8.63a limiting factor during AF. Various studies have concluded that the
Asparagine 13.93 � 0.31a 1.67 � 0.11b 8.51 � 0.40c minimum concentration of yeast assimilable nitrogen (YAN) for risk-free
Aspartic acid 37.43 � 0.26a 1.55 � 0.15b 5.85 � 0.44c
of slow or stuck fermentation was around 140 mg N/L in grape juices
Cysteine 5.17 � 0.49a 1.54 � 0.03b 2.25 � 0.12c
Glutamic acid 85.38 � 7.60a 4.39 � 0.10b 17.97 � 1.16c (Bell & Henschke, 2005). Despite the low YAN concentration (119.68
Glycine 3.96 � 0.14a 2.00 � 0.14b 7.74 � 0.09c N/L) in raw dragon fruit juice, AF was completed in both control and PT
Histidine 12.83 � 0.91a 1.86 � 0.18b 11.80 � 0.61a samples, indicating T. delbrueckii is a low nitrogen demanding yeast. The
Hydroxyproline 89.64 � 0.62a 3.31 � 0.27b 13.75 � 0.80c threshold of YAN requirement is largely strain dependent, even within
Isoleucine 5.50 � 0.64a 1.23 � 0.14b 3.35 � 0.33c
Leucine 32.37 � 1.07a 3.98 � 0.02b 13.38 � 1.18c
S. cerevisiae species, low nitrogen demanding strains could complete AF
Lysine 26.03 � 0.19c 4.05 � 0.19b 15.27 � 0.31c in media with YAN as low as 60 N mg/L (Lemos Junior et al., 2017),
Methionine 9.70 � 0.46a 1.69 � 0.05b 5.19 � 0.44c although the rate of AF was affected.
Ornithine 2.42 � 0.09a 1.55 � 0.03b 5.53 � 0.17c Pectinase pre-treatment significantly affected the nitrogen meta­
Phenylalanine 38.58 � 0.85a 3.64 � 0.47b 10.03 � 0.65c
bolism of T. delbrueckii, which decreased the utilization rate from 89.6%
Phosphorthanolamine 7.23 � 0.64a 0.436 � 0.537 �
0.020b 0.039c to 50.8% (Table 2). Few studies are available on the nitrogen meta­
Phosphoserine 4.93 � 0.33a 4.01 � 0.05b 4.88 � 0.58a bolism of non-Saccharomyces yeast or the effect of enzyme treatment.
Proline 184.20 � 20.07 � 1.05b 110.88 � For S. cerevisiae, the ability to metabolize nitrogen could be affected by
5.97a 3.83c adverse conditions. Beltran, Rozes, Mas, and Guillamon (2007) reported
Sarcosine 52.85 � 3.38a 6.89 � 0.31b 35.92 � 3.46c
Serine 12.62 � 1.03a 1.26 � 0.02b 3.60 � 0.21c
that under low fermentation temperature, the activity of some perme­
Taurine 19.25 � 1.42a 1.41 � 0.12b 1.18 � 0.13b ases was impaired due to the decrease of plasma membrane fluidity. It
Threonine 10.92 � 0.29a 1.20 � 0.15b 3.61 � 0.30c seems that pectinase either caused structural changes to yeast cells or
Tryptophan 7.68 � 0.34a 0.00 � 0.00b 3.92 � 0.21c affected its gene expression. Further studies on yeast cell structure and
Tyrosine 18.05 � 0.43a 2.96 � 0.13b 8.82 � 0.33c
the regulation of nitrogen metabolism at the genetic level are required to
Urea 0.00 � 0.00a 1.15 � 0.07b 4.06 � 0.38c
Valine 11.97 � 0.22a 1.22 � 0.06b 3.25 � 0.30c understand the potential effect.
β-aminoisobutyric acid 49.09 � 4.13a 44.77 � 1.53a 42.46 � 0.42a
ϒ-aminobutyric acid 25.52 � 0.85a 24.44 � 0.40a 37.23 � 1.91b 3.4. Effect of pectinase treatment on volatile aroma compounds
P
N (mg N/L) 119.68 13.60 58.92

Pectinex, pectic enzyme.
a, b,c, d
: Statistical analysis ANOVA (n ¼ 3) at 95% confidence level with the same
letter indicating no significant difference.
enzymatic breakdown of polygalacturonic acid backbones. Thus, the
concentration of D-galacturonic acid could be used as an indicator of
pectinase activities. The absence of galacturonic acid in control samples
suggested the lack of pectinase activities in either red dragon fruit juice
or the yeast strain T. delbrueckii. Approximately 60% of D-galacturonic
acid in PT sample was produced on day 0, right after 1-h Pectinex
treatment; the rest was accumulated during fermentation, indicating the
persistence of pectinase activity throughout AF (Fig. 2A).
3.2. Effect of pectinase treatment on juice/wine yield and viscosity
Significant increases in yield and decrease in viscosity were observed
in PT samples (Table 1). Raw red dragon fruit juice had a relatively high
viscosity (9.4 cP) due to the gelatinous pectic substances that stayed
linked with pulp. The pectin fraction in dragon fruits has a low degree of
methoxylation (DM) value (5–39%) (Liaotrakoon et al., 2013), which
gelates in the presence of a high amount of sugar at low pH. One-hour
treatment with pectinase did not produce significant effects on juice
yield or viscosity immediately. Similar findings in previous studies
showed that application of pectolytic and cellulolytic enzymes in dragon
fruit juice resulted in surprisingly low reduction in viscosity (Bellec
et al., 2006; Truong & Dang, 2016). Upon fermentation, there was a
significant decrease in viscosity and increase in yield in PT samples. The
transformation from fruit juice into wine involves the major change of
sugars into ethanol which effectively reduced the viscosity of the media.
Pectinex treatment seemed to have a synergistic effect on wine yield. PT
sample had 16% higher yield and 1.5 cP lower in viscosity than control
Significant increases in the level of total volatile compounds were
observed in red dragon fruit wines after fermentation, in particular,
higher alcohols, esters, and terpenes (Table 3). The levels of fatty acids
and aldehydes, which are normally associated with acidic, fatty or green
aromas in the juice, were reduced. PT sample contained slightly less
amounts of total volatile aroma compounds than control sample based
on GC-FID analysis (Table 3). This difference was attributed to the lower
production of higher alcohols in PT sample, especially isoamyl alcohol
and 2-phenylethyl alcohol. Leucine and phenylalanine are the amino
acid precursors for these two volatile compounds, respectively. Their
relatively low utilization rate in PT sample (Table 2) may explain the
low production of these two volatile compounds. On the other hand, PT
sample contained significantly higher levels of esters than control
samples (more than 2-fold of the total FID peak area), in particular, ethyl
acetate, 2-phenyethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl
decanoate and ethyl dodecanoate, all of which impart floral and fruity
flavors. Yeasts form higher alcohols and esters through Ehrlich pathway
and transamination from amino acids (Bell & Henschke, 2005). Many
studies have shown a significant correlation between YAN and volatile
compounds production; however, recent studies suggest the main role of
the catabolism of most consumed amino acids is to supply nitrogen for
the synthesis of proteinogenic amino acids (Gobert, Tourdot-Marechal,
Sparrow, Morge, & Alexandre, 2019). The carbon skeleton from amino
acids contributes to only a fraction of the volatile compounds produc­
tion, depending on the level of YAN available.
Terpenes are present in small amounts in the raw juice, composed
mainly of linalool. After fermentation, β-myrcene and longifolene con­
centration increased, while the level of linalool and α-terpineol
decreased in both samples. In grapes, terpenes/terpene alcohols are
typically bound to sugars. The release of free volatile terpenes involves
enzyme hydrolysis by β-D-glucosidase, which is not produced by many
S. cerevisiae strains. T. delbrueckii is well known for its ability to produce

5
X. Jiang et al. LWT 132 (2020) 109929

Table 3
Major volatile compounds (GC-FID peak area x 106) and their relative peak areas (RPA) identified in red dragon fruit juice and wines fermented with T. delbrueckii
without (Control) and with Pectinex pre-treatment (Pectinex).
Identified volatile compounds LRI Day 0 Day 14 Organoleptic
Peak area RPA Control Pectinex
(%) Peak area RPA Peak area RPA
(%) (%)
Acid
Acetic acid 1453 1.27 � 0.14a 0.93 4.33 � 0.37b 0.10 5.90 � 0.68c 0.18 Acidic, pungent, vinegar-like
Hexanoic acid 1836 20.00 � 14.60 0.00 � 0.00b 0.00 0.00 � 0.00b 0.00 Cheesy, fruity, fatty
1.82a
Octanoic acid 2052 9.71 � 1.27a 7.08 2.86 � 0.17b 0.07 2.32 � 0.22c 0.07 Acidic, cheesy, fruity
Nonanoic acid 2160 1.29 � 0.42a 0.94 0.26 � 0.01b 0.01 0.00 � 0.00c 0.00 Waxy, cheesy, dairy
Subtotal 32.28 23.55 7.46 0.17 8.22 0.24
Alcohol
1-Propanol 1025 0.00 � 0.00a 0.00 0.00 � 0.00a 0.00 5.92 � 0.50b 0.18 Alcoholic, fruity, musty
Isoamyl alcohol 1229 0.00 � 0.00a 0.00 172.4 � 9.7b 3.91 84.04 � 2.51 Alcoholic, whiskey, fruity, banana
4.09c
1-Hexanol 1371 79.24 � 57.81 4.06 � 0.12b 0.09 0.00 � 0.00c 0.00 Fusel, fruity, alcoholic, sweet, green
5.06a
2-Phenylethyl alcohol 2005 0.00 � 0.00a 0.00 172.2 � 3.91 145.3 � 4.6b 4.33 Floral, rose, dried rose
17.1b
Subtotal 79.24 57.81 348.63 97.85 235.20 94.60
Aldehydes
b b
Hexanal 1085 11.60 � 8.46 0.00 � 0.00 0.00 0.00 � 0.00 0.00 Fresh, green, grass leafy
1.89a
2-Hexenal 1222 2.01 � 0.09a 1.46 0.00 � 0.00 b
0.00 0.00 � 0.00b
0.00 Fruity, green, leafy, vegetable
Benzaldehyde 1538 0.00 � 0.00a 0.00 1.90 � 0.14b 0.04 1.70 � 0.19b 0.05 Almond like
Phenyl acetaldehyde 1651 2.59 � 0.23a 1.89 0.00 � 0.00b 0.00 1.30 � 0.13c 0.04 Floral, sweet
2,4-Dimethyl benzaldehyde 1838 0.00 � 0.00a 0.00 0.26 � 0.01b 0.01 0.72 � 0.12c 0.02 Cherry, almond, spicy, vanilla
Subtotal 16.19 11.81 2.16 0.05 3.72 0.11
Volatile compounds identified in this LRI Day 0 Day 14 Organoleptic
study Peak area RPA Control Pectinex
(%) Peak area RPA Peak area RPA
(%) (%)
Ketones
3-Hydroxy-2-butanone 1305 0.00 � 0.00a 0.00 1.14 � 0.08b 0.03 0.00 � 0.00a 0.00 Sweet, buttery, creamy, milky
Ethylidene acetone 1391 0.00 � 0.00a 0.00 0.74 � 0.06b 0.02 0.00 � 0.00a 0.00 Fruity, acetone, phenolic, fishy
Subtotal 0.00 1.89 0.04 0.00
Ester
Ethyl acetate 968 0.00 � 0.00a 0.00 15.88 � 0.36 24.34 � 0.73 Ethereal, fruity, solventy
0.76b 0.75c
Isoamyl acetate 1115 0.00 � 0.00a 0.00 19.06 � 0.43 13.19 � 0.39 Sweet, banana, ripe
1.58b 1.44c
Methyl hexanoate 1174 2.16 � 0.23a 1.58 0.00 � 0.00b 0.00 0.00 � 0.00b 0.00 Fruity, pineapple, ethereal
Ethyl hexanoate 1215 0.00 � 0.00a 0.00 0.00 � 0.00a 0.00 26.03 � 0.78 Sweet, fruity, pineapple, green
0.26b
Hexyl acetate 1262 0.00 � 0.00a 0.00 5.69 � 0.58b 0.13 0.00 � 0.00a 0.00 Fruity, green, apple, banana, sweet
Ethyl heptanoate 1320 0.00 � 0.00a 0.00 0.52 � 0.01b 0.01 0.00 � 0.00a 0.00 Fruity, pineapple, cognac, rummy,
winey
Ethyl octanoate 1425 0.22 � 0.08a 0.16 16.58 � 0.38 39.60 � 1.18 Fruity, pineapple, creamy, dairy
0.47b 7.37c
a
Ethyl nonanoate 1523 0.00 � 0.00 0.00 0.00 � 0.00a 0.00 0.00 � 0.00a 0.00 Sweet, fruity, pear
Ethyl decanoate 1627 0.00 � 0.00a 0.00 5.87 � 0.35b 0.13 30.00 � 0.89 Sweet, waxy, apple
1.37c
Diethyl succinate 1670 0.00 � 0.00a 0.00 0.24 � 0.03b 0.01 0.36 � 0.02c 0.01 Fruity, cooked apple, passion fruit
Ethyl 9-decenoate 1681 0.00 � 0.00a 0.00 0.28 � 0.04b 0.01 0.27 � 0.02b 0.01 Fruity, fatty
Ethyl phenylacetate 1784 0.00 � 0.00a 0.00 0.21 � 0.03b 0.00 0.00 � 0.00a Sweet, floral, rose, balsamic, cocoa
2-Phenethyl acetate 1816 0.00 � 0.00a 0.00 1.55 � 0.12b 0.04 2.54 � 0.26c 0.08 Floral, rose, sweet, honey, fruity,
tropical
a b
Ethyl dodecanoate 1834 0.00 � 0.00 0.00 5.88 � 0.41 0.13 12.93 � 0.39 Sweet, waxy, fruity, apple, grape,
1.44c brandy
Isobutyl Pentadecanoate 1855 0.00 � 0.00a 0.00 0.34 � 0.00b 0.01 0.75 � 0.00c 0.02 Fatty
Phenethyl propionate 1884 0.00 � 0.00a 0.00 0.43 � 0.08b 0.01 0.22 � 0.04c 0.01 Floral, rose, fruity, honey, balsamic
Ethyl myristate 2042 0.00 � 0.00a 0.00 2.21 � 0.27b 0.05 7.60 � 0.67c 0.23 Sweet, waxy, violet
Subtotal 2.37 1.73 74.73 1.70 157.82 4.71
Volatile compounds identified in this LRI Day 0 Day 14 Organoleptic
study Peak area RPA Control Pectinex
(%) Peak area RPA Peak area RPA
(%) (%)
Terpene derivatives
β-Myrcene 1140 0.77 � 0.09a 1.58 2.74 � 0.33b 0.06 2.64 � 0.22b 0.08 Peppery, terpenic, spicy, balsamic
D-Limonene 1170 0.00 � 0.00a 0.00 1.40 � 0.09b 0.03 0.00 � 0.00a 0.00 Citrus, orange, fresh, sweet
Linalool 1539 4.96 � 0.41a 0.00 0.77 � 0.07b 0.02 0.57 � 0.02c 0.02 Citrus, floral, sweet, rose
Longifolene 1554 0.94 � 0.07a 0.00 3.47 � 0.42b 0.08 8.21 � 0.36c 0.24 Sweet, woody, rose
α-Terpineol 1697 0.30 � 0.02a 0.16 0.20 � 0.03b 0.00 0.00 � 0.00c 0.00 Pine, terpenic, lilac, citrus, woody,
floral
Subtotal 6.98 1.73 8.59 0.19 11.4 0.34
(continued on next page)

6
X. Jiang et al. LWT 132 (2020) 109929

Table 3 (continued )
Others
Styrene 1249 0.00 � 0.00a 0.00 � 0.00a 8.90 � 1.22b 0.27 Sweet, plastic, balsamic

Subtotal 137.06 100.00 443.45 100.00 416.39 100.00

LRI: liner retention index determined in the DB-FFAP column.

Organoleptic descriptors were obtained from http://www.thegoodscentscompany.com (accessed 8 October 2018).
Pectinex, pectic enzyme.
a, b,c, d
Statistical analysis ANOVA (n ¼ 3) at 95% confidence level with the same letter indicating no significant difference.

and selection of yeast strains, could have a more pronounced influence

Table 4 than the application of pectinase enzyme. Optimization of enzyme
Total phenolic content (TPC), total antioxidant capacity (TAC), betacyanin treatment in terms of enzymes composition, purity, application dosage
content (BC) and CIELab color parameters of red dragon fruit wines fermented and conditions may be required to achieve more plentiful aroma profile.
with T. delbrueckii without (Control) and with Pectinex pre-treatment (Pectinex).
Day 0 Day 14

Control Pectinex Control Pectinex 3.5. Effect of pectinase treatment on BC, TPC, TAC and color stability
TPC (mg/L 340.71 � 346.58 � 275.34 � 302.83 �
GEA) 19.67a 16.68a 8.18b 8.78c Betacyanin is the major betalain pigment responsible for the char­
TAC (mM TE) 7.81 � 0.57a 8.17 � 0.17a 9.99 � 0.68b 9.44 � 0.34b acteristic purple red color in the flesh and skin of red dragon fruit. In raw
BC (mg/L BE) 179.35 � 164.08 � 112.24 � 75.88 � red dragon fruit juice, the BC, TPC and TAC values were 179.35 BE mg/
8.92a 0.61b 1.98c 3.79d L, 340.71 GAE mg/L and 7.81 TE mmol/L, respectively (Table 4). These
Color
L* 34.13 � 0.04a 33.04 � 0.01b 35.57 � 33.00 �
values were lower than that of previous studies (Stintzing et al., 2003;
0.05c 0.28b Wu et al., 2006), which could be due to different extraction methods
a* 76.00 � 0.06a 73.60 � 0.01b 79.45 � 76.86 � 0.35c used, as well as the heat treatment.
0.06c Upon fermentation with T. delbrueckii, the evolution of BC (Fig. 2B),
b* 5.60 � 0.09a 11.14 � 0.13b 8.52 � 11.82 �
TPC (Fig. 2C) and TAC (Fig. 2D) in both control and PT samples followed
0.41c 0.64d
C* 76.20 � 0.07 a
74.44 � 0.01 b
79.90 � 77.76 � similar patterns. BC decreased consistently throughout the fermentation
0.10c 0.42d process, while TPC decreased in the early stage and increased slightly
h� 4.21 � 0.05a 8.61 � 0.11b 353.9 � 0.3c 351.3 � from day 10 until the end of the fermentation. Overall, there was 37%
0.44d and 58% decrease in BC; 19% and 11% decrease in TPC in control and
Pectinex, pectic enzyme. PT samples respectively. TAC increased sharply on day 2 and then
GEA, gallic acid equivalent; TE, trolox equivalent; BC, betacyanin equivalent. fluctuated until the end of AF with slight increase than Day 0 for both
L*, corresponds to lightness of sample with value ranging from 0 (for pure black) treatments. Application of Pectinex did not seem to significantly influ­
to 100 (for pure white); a*, corresponds to redness when positive and greenness ence TAC either before or after fermentation.
when negative; b*, corresponds to yellowness when positive and blueness when In red dragon fruit juice, the major bioactive compounds present are
negative; C*, chroma, C* ¼ (a*2 þ b*2)1/2; h� , hue angle, h� ¼ arctan (b*/a*),
betacyanins, flavonoids and phenolic acids, in which betacyanins
respectively.
a, b, c, d contribute to the highest antioxidant capacity (Tenore, Novellino, &
Statistical analysis ANOVA (n ¼ 3) at 95% confidence level with the same
Basile, 2012). The decrease of BC could be due to the presence of
letter indicating no significant difference.
β-glucosidase activity in T. delbrueckii and Pectinex. This is because
β-glucosidase could hydrolyze the glycosidic bond in glucosylated
β-D-glucosidase (Cus & Jenko, 2013). There is a significantly higher
betacynins (such as betanin or phyllocactin) and release the less stable
amount of terpenes, especially longifolene found in PT sample which
betanidin (Gengatharan, Dykes, & Choo, 2015). There is a strong cor­
could be due to the presence of β-D-glucosidase activity present in the
relation between BC and TPC for both control and PT samples (r ¼ 0.97
enzyme preparation. Ward, Qin, Dhanjoon, Ye, and Singh (2006) re­
for control and r ¼ 0.90 for PT sample), which could be attributed to the
ported that besides pectolytic enzymes, Pectinex® Ultra SP-L also con­
phenolic structure present in betacyanins that contributed to TPC, as
tains a small amount of hemicellulases and cellulases.
well as possible synergistic interactions between betanins and phenolic
A study on the effect of applying pectinase enzyme to Cabernet
compounds (Zainoldin & Baba, 2009). Despite the greater loss in BC, PT
Gernischt dry red wine during maceration showed similar results to the
sample contained higher final TPC, which indicated greater release of
current study with more terpenes and phenylethyl alcohol, but less total
flavonoids or phenolic acids via enzyme hydrolysis.
higher alcohols produced (Sun, Hu, Zhang, Zhu, & Tao, 2018). However,
Contrary to popular belief, pectinase treatment does not always show
contradictory results on the effect of pectinase treatment on the aroma
significant impacts on TPC or TAC of wine and the effect on the release
compound production in fruit wines or alcoholic beverages have also
of phenolic compounds depends largely on the food matrix, polyphenol
been reported. Mango wine treated with pectinase showed increased
composition, application condition as well as enzyme characteristics.
production of higher alcohols including isoamyl alcohol, 2-phenylethyl
Addition of pectolytic enzyme during maceration generally resulted in
alcohol, n-propanol, n-butanol and harmful methanol, which corre­
higher levels of TPC or TAC in fruit juice or pomace; however, this effect
sponded to higher sensory scores (Reddy & Reddy, 2009). Zhang,
often became insignificant after fermentation and the subsequent
Woodams, and Hang (2011) found little effect on the concentration of
maturation of wine (Alpeza et al., 2017; Ma et al., 2018; Samoticha
most of higher alcohols in pectinase treated apple spirits, but significant
et al., 2017). In grape wine, degradation of fruit cell walls induced by
increases in methanol. Another study on the effect of pectolytic enzymes
enzyme addition may facilitate the extraction of higher molecular
treatment and type of yeast on the chemical properties of Solaris white
weight tannins, while the concentrations of lower molecular weight
wine (Samoticha et al., 2017) concluded that the type of yeast exerted a
compounds such as anthocyanins are affected by both their extraction
significant influence on total volatile compounds while enzyme treat­
from grapes and subsequent reactions in wine. The difference in
ment did not make any significant difference. These findings showed
phenolic composition in samples arising from easier extraction of some
that the volatile compound composition of fruit wine is affected by a
phenolic compounds than others by enzymes may be the major
variety of factors, some of which such as type of fruit, maturity of fruit
contributor to the inconsistency observed between TPC and TAC of wine

7
X. Jiang et al.
(Bagger-Jorgensen & Meyer, 2004). The concentration of phenolics in
wine depends not only on the maceration process but also the level of
degradation arising from hydrolysis and oxidation as well as yeast ab­
sorption. Detailed analysis on the composition of phenolic compounds in
juice and wine may help explain the observed trend in TAC and TPC
during enzyme hydrolysis and fermentation.
Significant decrease in color intensity was observed upon fermen­
tation and application of pectinase (Table 4). The L* and a* values
decreased upon enzyme treatment both before and after fermentation,
indicating decreases in lightness and redness compared to control
sample, which correlated with the decrease of betacyanin pigment.
Degradation of betacyanins upon enzyme treatment resulted in the
production of orange, yellow or colorless compounds (Herbach, Rohe,
Stintzing, & Carle, 2006). Upon subsequent fermentation, PT sample
showed increase in purple blue hue, indicated by the shifting of b* and h�
value which could be brought about by the release of dark colored
pigments from red dragon fruit pulp or seed.
4. Conclusions
Pectinase pre-treatment displayed some promising results on red
dragon fruit wine fermented with T. delbrueckii including increased wine
yield (by 16%), more plentiful flavor/aroma profile (higher levels of
esters and terpenes, lower levels of higher alcohols) as well as higher
total phenolic content. On the other hand, application of pectinase
caused higher production of acetic acid and significant impairment on
the yeast’s ability to metabolize nitrogen containing compounds. Pec­
tinase treated sample also showed a greater loss of betacyanin pigment
and color intensity. This study suggested that with further optimization
of enzyme treatment there is a great potential of red dragon fruit wine
fermented with T. delbrueckii as an exotic alternative wine.
CRediT authorship contribution statement
Xiaohui Jiang: Writing - original draft, Conceptualization, Meth­
odology, Formal analysis, Data curation, Writing - original draft. Yuyun
Lu: Writing - original draft, Conceptualization, Methodology, Formal
analysis, Data curation, Writing - original draft, Writing - review &
editing. Shao Quan Liu: Conceptualization, Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
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