Research article Institute of Brewing & Distilling

Received: 4 July 2016 Revised: 4 January 2017 Accepted: 5 January 2017 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jib.412

Enhancement of volatile aromatic compounds

in black raspberry wines via enzymatic
treatment
Byoung-Ho Kim and Seung K. Park*
Robus coreanus Miquel is widely used in the production of Korean black raspberry (KBR) wine owing to its health benefits
and commercial value. The effects of three different commercially available glycosidase enzymes on the volatile compounds in
KBR wines were investigated with large-scale fermentation to develop a high-flavour-quality wine. Volatile aroma compounds
from the wines were analysed using headspace-solid phase micro-extraction-gas chromatography–mass spectrometry
(HS-SPME-GC–MS) and sensory evaluations were performed to evaluate the flavour characteristics. KBR wines treated with
commercial enzymes yielded high concentrations of terpenes and esters compared with the control wine because the odourless
non-volatile glycosides in KBR wines were converted to their corresponding free forms by the enzymes. HS-SPME-GC–MS-SIM
analysis showed that the primary monoterpene compounds in KBR wines treated by the enzymes were myrtenol, linalool,
citronellol and a significant quantity of compounds such as 2-phenyl ethanol and ethyl benzoate, which contributed to the
flavour of KBR wine, as determined by GC-FID. KBR wines treated with the enzymes exhibited different sensory characteristics
from the control wine owing to higher intensity of floral and fruity aromas. KBR wine treated with CYTOLASE PCL5 on a large scale
exhibited the highest sensory preference. Copyright © 2017 The Institute of Brewing & Distilling

Keywords: black raspberry wine; aroma analysis; commercial enzyme; HS-SPME-GC–MS-SIM; large-scale fermentation

Introduction widely used on a large scale for aroma enhancement, clarification

Robus coreanus Miquel is currently being grown in various regions
of Asia, including Korea, China and Japan. The commercial value of
Robus coreanus Miquel is very high owing to its health benefits
(1–4). In our previous report (5), we selected a particular yeast
strain to produce high-quality Korean black raspberry (KBR) wine
using chemical and sensory analysis. We further considered the
use of commercial enzymes to enhance the aroma of KBR wine
fermented with the selected yeast strain on a large scale.
Many reports have been published with regard to enzyme
treatment in wines (6–16); however, few studies have reported
the aromatic enhancement of KBR wine on a large scale using
various commercial enzymes. The typical aroma of wine is
primarily due to volatile compounds derived from grapes,
including monoterpenes, norisoprenoids, aliphatics and phenolic
compounds; other factors that can affect the aroma of wine
include geographical and cultural factors and winemaking
techniques (6). The noted volatile compounds possess pleasant
floral and fruity aromas, with very low perception thresholds. The
derived flavour compounds are present in the wine as free-form
and odourless non-volatile glycosides. The aglycone moieties of
glycosides are known to be partly bound to sugar molecules such
as rhamnose-glucose, arabinose-glucose and apiose-glucose (7).
These glycosides represent a potential flavour reservoir; the
volatile compounds from glycosides can be released by acids or
through glycosidase enzyme hydrolysis (8). Several exogenous
enzymes from fungi have been developed to liberate the aromatic
compounds in wines and are widely used in the winemaking
process (9). Commercial enzymes contain high levels of
pectinolytic, cellulolytic and β-glucosidic activity and are being
and colour extraction of tea, juice and fruit. The costs are
reasonable for industrial purposes.
In this study, headspace-solid phase micro-extraction-gas
chromatography–mass spectrometry (HS-SPME-GC–MS) was
applied to determine the total quantity of terpenes and other
volatile compounds in young black raspberry wine with and
without enzymatic treatment. HS-SPME-GC–MS is the method
of choice for the analysis of bound aromatics in wines because
the process does not use solvent and, is rapid, convenient,
highly reproducible and highly sensitive (10). GC–MS analysis
with selected ion monitoring (SIM) was adapted to analyse the
aromas in KBR wine at a low level owing to its high sensitivity
and selectivity.
The aim of this study was to investigate the effects of
commercial enzymes on the aromatic enhancement of black
raspberry wine grown in Korea in large-scale production.
Materials and methods
Black raspberry wine production on a large scale
Korean black raspberry wines were produced according to the
standard KBR winemaking processes established by the HiteJinro
* Correspondence to: Seung K. Park, Department of Food Science and
Biotechnology, KyungHee University, Yongin-si, 446–701, Republic of Korea.
E-mail: skpark@khu.ac.kr
Department of Food Science and Biotechnology, KyungHee University,
Yongin-si 446-701, Republic of Korea

J. Inst. Brew. 2017 Copyright © 2017 The Institute of Brewing & Distilling

Institute of Brewing & Distilling
Brewery Co. Ltd. The commercial dried yeast (M1, Lallemand,
Montreal, QC, Canada) selected as the target yeast strain in our
previous report (5) was used for large-scale production of KBR
wine. The activated yeast was inoculated into 200 kg of defrosted
raspberry must and was incubated for 24 h at 25°C to generate the
starter culture. The inoculum concentration was 200 mg (dry
weight) kg 1 raspberry must. After production of the starter
cultures for 3 days at 25°C, 800 kg of the thawed must and 80 kg
(additional mass of sucrose used to adjust the sugar concentration
to 24° Brix) of sucrose were added to a 1000 kg fermentation tank.
The raspberry must was fermented for 7 days at 25°C. At the end of
the fermentation process, the wine was clarified through the
addition of 2 g L 1 bentonite and the resulting wine was stored
at 25°C in an aging tank.
Assay of β-glucosidase activity
The assay of β-glucosidase activity was measured in modification
of the method reported by Lee et al. (11). β-Glucosidase activity
was assayed by measuring the amount of P-nitrophenol
hydrolysed from p-nitrophenyl-β-D-glucoside as substrate. The
procedure of enzyme assay reaction is as follows: 50 μL of
each commercial enzyme solution was added into Eppendrof test
tubes and incubated for 15 min in a water bath at 37°C with 150 μL
of 0.1 M phosphate buffer (pH 6.0) and 100 μL of 2 mM
p-nitrophenyl-β-D-glucoside dissolved in buffer. The reaction was
stopped by adding 400 μL of 0.5 M NaOH solution. The mixture
was centrifuged for 10 min at 1500 g and the absorbance
measured at 400 nm using a spectrophotometer. One unit of
β-glucosidase is the amount of enzyme that catalyses the
hydrolysis of 1.0 μmol substrate per minute at pH 7.0.
Enzyme treatment
In order to release bound aromas in the KBR wines, three
commercial enzyme formulations were tested after the
fermentation process because β-glucosidase activity was
inhibited by the exoglycosidase activitie of glucose (12). The
commercial enzyme formulation CYTOLASE PCL5 (DSM Food
Specialities, USA) produced from Aspergillus niger consisted of
a complex of enzymatic components including pectinase,
cellulase, hemicelluloses and β-glucosidase. The second enzyme,
AROMASE™ H2 (Amano Enzyme, Japan), produced from
Penicillium multicolour, is a kind of β-glucosidase that hydrolyses
di-glycosides such as β-primeverosidase. The third enzyme,
SUMIZYME AC (Shin Nihon Chemical, Japan), produced from A.
niger, is an enzyme complex consisting of cellulase, hemicellulase
and β-glucosidase. Conditions of the enzyme reaction were
established through a recommendation of enzyme suppliers
and were based on the KBR wine manufacturing process
at HiteJinro Brewery Co. Ltd. The measurement results of
β-glucosidase activity in three commercial enzymes were 1231.5
u g 1 for AROMASE™ H2, 89.2 u g 1 for CYTOLASE PCL5 and
65.4 u g 1 for SUMIZYME AC. AROMASE™ H2 showed the highest
β-glucosidase activity. Considering the manufacturing process
and the difference of β-glucosidase activity of three commercial
enzymes, the employed enzyme concentrations were 300 mg
L 1 for CYTOLASE PCL5 and SUMIZYME AC and 100 mg L 1
for AROMASE™ H2. The conditions of the enzymatic reaction
were pH 3.5 at 25°C for 30 days following the addition of
the enzyme.
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B.-H. Kim and S. K. Park
HS-SPME-GC–MS analysis
Analysis of the volatile compounds in KBR wines treated with
commercial enzymes was carried out using HS-SPME-GC–MS.
The KBR wine samples (5 mL) and 0.5 mL of a 50 mg L 1 solution
of 2-octenal (internal standard) were added to a 20 mL vial with a
silicone septum and aluminium cap (Supelco, Bellfonte, PA, USA).
Sample preparation was performed using a Gerstel SPME
Autosampler (MPS-3, Gerstel GmbH, Műlheiman der Ruhr,
Germany) with a 2 cm-long 50/30 μm DVB/CAR/PDMS fibre from
Supelco. The samples were pre-incubated for 10 min at 60°C and
the extraction was performed in the headspace of each vial for
30 min at 60°C. Desorption was performed in the injector of
an Agilent 5975 GC/MS (Santa Clara, CA, USA) equipped with a
DB-5 fused silica capillary column (0.25 mm × 30 m i.d.,
0.25 μm film thickness). Helium was used as the carrier gas at a
total flow rate of 1 mL min 1. The oven was programmed to hold
a temperature of 45°C for 5 min, followed by ramping from 45 to
220°C at rate of 10°C min 1, then was held at 220°C for 10 min.
The mass spectrometer was operated in electron impact mode
with an ionization voltage of 70 eV, and an ion source
temperature of 230°C. Mass spectra were acquired by scanning
the mass range between 40 and 500 at an acquisition rate of
3.15 scans s 1.
Identification and quantification of volatile compounds
Among the volatile compounds in KBR wines, various terpenes
were identified by comparing their retention times and matching
their mass spectra with those of standards and other referenced
compounds in the Wiley library and NIST Mass Spectral Search
Program (ChemSW Inc., NIST 98 Version Database) connected to
the Agilent 5975 mass spectrometer. Quantification was achieved
by comparing the GC retention times with those of authentic
standards from Sigma-Aldrich and with GC–MS selective ion
monitoring mode. Among the volatile compounds in KBR
wines, terpenes were present at very low-levels; therefore, the
GC–MS-SIM mode is more effective than the GC–MS-full-scan
mode for the quantification of terpenes. The GC–MS-SIM mode
was more than 10–100 times more sensitive than that of the
GC–MS-full scan mode. Because unwanted ions were being
filtered, selectivity was greatly enhanced, providing an additional
tool to eliminate difficult interferences. For SIM mode, two
characteristic ions were selected for each compounds and were
scanned using corresponding time windows, with dwell times
(the time spent looking at each mass) in the range of 150 ms per
ion. The quantification ions for SIM mode are presented in
Table 1. The quantification standards, representing several major
terpenes of KBR wine, were purchased from Sigma-Aldrich:
ρ-cymene, β-linalool, terpine-4-ol, ρ-cymene-8-ol, myrtenol,
verbenone, citronellol, geraniol and 2-octenal as an internal
standard. Standard curves were developed using HS-SPME
sampling methods (60°C for 30 min, DVB/CAR/PDMS fibre) with
subsequent injection into the GC–MS-SIM using the conditions
described above. The peak area of each standard target ion
relative to the peak area of the 2-octenal internal standard was
plotted against the ratio of the standard to 2-octenal in order to
develop a standard curve. Linear regression equations were used
to calculate the concentration (mg L 1) of each compound in all
samples. Alcohols and esters found in the KBR wines were
quantified using chemical standards purchased from Sigma-
Aldrich: 1-propanol, 2-methyl-1-propanol, 1-butanol, 3-methyl-1-
J. Inst. Brew. 2017
Volatiles in black raspberry wine via enzymatic treatment
Institute of Brewing & Distilling

Table 1. Selected ion groups of terpene compounds for Results and discussion
GC–MS-SIM mode analysis
Effects of various commercial enzymes on the volatile
Compounds Dwell time (min) Selected ions (m/z) compounds in KBR wines
ρ-Cymene 10.00 91, 119 In our previous report (5), 67 volatile compounds were identified
2-Octenal (IS) 11.50 55, 70 from KBR wine via HS-SPME-GC–MS. Of these, eight terpenes, four
β-Linalool 12.50 55, 71 esters and five alcohols, postulated to contribute to wine aroma,
Terpine-4-ol 14.05 71, 93 were quantified by GC-FID and HS-SPME-GC–MS in order to verify
ρ-Cymene-8-ol 14.20 91, 117 the effects of various commercial enzymes (Table 2). To study the
Myrtenol 14.30 79, 91 effect of glycosidase enzyme treatment on wine aroma, the
Verbenone 14.65 91, 107 compounds present as glycosidically bound forms in wine were
Citronellol 14.80 55, 69 taken into consideration. The enzymatic enhancement of terpenes
Geraniol 15.00 69, 93 was important for the production of high-quality wine because
IS, Internal standard. terpenes positively affect wine aroma. In our previous report (5),
the eight terpenes ρ-cymene, β-linalool, terpine-4-ol, ρ-cymene-8-
ol, myrtenol, verbenone, citronellol and geraniol were the most
common in KBR wine. Different commercial enzymes possessing
butanol, 2-phenyl ethanol, ethyl acetate, ethyl lactate, ethyl β-glucosidase activity release aromatic compounds through
benzoate and 2-phenylethyl acetate. These compounds were cleavage of the glycosidic bond of glycoside. ρ-Cymene is a
analysed by GC-FID equipped with a DB-WAX fused silica capillary monoterpene with a fresh and citrus odour (18); as shown in
column (0.32 mm × 30 m i.d., 0.25 μm film thickness) after Table 2, the level of ρ-cymene in KBR wine treated by AROMASE™
distillation. A 2 μL sample was injected in splitless mode. The H2 was higher than that of wine treated with CYTOLASE PCL5 or
temperature programme held a temperature of 45°C for 1 min, SUMIZYME AC. β-Linalool is a terpene alcohol found in many
followed by ramping to 230°C at a rate of 20°C min 1, before flower and spice plants and possesses a flowery, lavender-like
holding the isotherm for 10 min. Helium was used as the carrier odour (19); a slight increase in concentration was observed for
gas (1 mL min 1). The temperature of the injector and detector KBR wines treated with the three commercial enzymes compared
was 250°C. Identification of the compounds detected by GC-FID with the control wine. Terpine-4-ol, also known as 4-terpineol, is a
analysis was performed through comparison with the authentic terpene alcohol with a characteristic pine-tree-like odour (20,21).
standards and published data. Quantification of the compounds Terpine-4-ol had the greatest effect on wine owing to its odour
was performed from the GC-peak areas relative to the GC-peak threshold (0.005 mg L 1), exhibiting the greatest increase in KBR
area of the internal standard. The results are expressed as the wine treated with AROMASE™ H2. ρ-Cymene-8-ol is a terpene
means ± standard deviation of three replicates. alcohol with an herbaceous celery-like odour (22). The level of ρ-
cymene-8-ol in SUMIZYME AC treated-wine was lower than that
in wine treated with AROMASE™ H2 or CYTOLASE PCL5. Myrtenol
is a monoterpene alcohol with a woody-like, minty odour (23).
Sensory analysis Among the various terpenes in KBR wine, myrtenol was detected
Wine samples treated by the three different commercial enzymes in the highest quantities. The most predominant terpene in KBR
and the control wine were analysed in triplicate by 25 trained wine was myrtenol. When comparing the terpene groups in black
panellists at the HiteJinro Brewery Co. Ltd, in addition to raspberry wines with those reported in the literature (24), the
experienced tasters in the KBR wine manufacturing field in Korea. compositions of terpenes in KBR wine were different, the reasons
A 30 mL sample of each wine was evaluated in a wine tasting glass being differences in the quantity and composition of terpenes,
covered with a glass Petri dish at 12°C. The seven flavour attributes reflective of different varieties of plant, climates, soil and degrees
(sweet, fruity, floral, citrusy, herb, waxy and off-odour) were scored of ripeness (12). After enzymatic hydrolysis, the detected quantities
from 0 (no flavour) to 5 (strong flavour) in intervals of 0.5. The of mytenol were ordered as follows: AROMASE™ H2 > CYTOLASE
mean scores were obtained and plotted as a polygonal graph. PCL5 > SUMIZYME AC. Verbenone is an organic compound
The sensory evaluation was performed using a quantitative classified as a terpene with a pleasant characteristic odour (22).
descriptive methodology (17). The amount of verbenone released with AROMASE™ H2 was higher
than that of wine treated with CYTOLASE PCL5 or SUMIZYME AC.
Citronellol, or dihydrogeraniol, is an acyclic monoterpenoid with
a green lemon odour (19). The quantity of citronellol increased
Statistical analysis slightly after treatment with the three commercial enzymes
The experimental results are reported as means ± SD of compared with the control wine. Geraniol is a monoterpene
measurements performed in triplicate. Means, relative standard alcohol with a floral and rose-like odour (19) and is used to impart
deviations (RSD) and linear regressions (standard curves) were flavours such as peach, raspberry, grapefruit and blueberry. As
calculated in Excel (Microsoft, Redmond, WA, USA). Principal shown in Table 2, the level of geraniol in KBR wine treated with
component analysis (PCA) was performed using SPSS version 20 AROMASE™ H2 was higher than that of wine treated with
software (SPSS Inc., Chicago, IL, USA). Statistical analyses were CYTOLASE PCL5 or SUMIZYME AC. Among the three commercial
calculated using analysis of variance (ANOVA) and a significance enzymes, AROMASE™ H2 significantly enhanced the release of
threshold of p < 0.05 was applied to correct for multiple tests. terpenes such as ρ-cymene, β-linalool, terpine-4-ol, ρ-cymene-8-
Significant differences between means were determined using ol, myrtenol, verbenone, citronellol and geraniol owing to its ability
Duncan’s multiple range tests. to hydrolyse bound forms. The total terpene content (119.0 mg

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 B.-H. Kim and S. K. Park
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Table 2. Concentration (mg L 1) of volatile compounds in KBR wines treated with different enzymes

Compounds Control wine CYTOLASE PCL5 SUMIZYMEAC AROMASE™ H2 Odour description

Terpenes ρ-Cymene 6.3 ± 0.23 7.1 ± 0.77 6.1 ± 0.14 8.5 ± 0.43 Weak, citrus odoura
β-Linalool 10.5 ± 0.96 12.5 ± 1.02 11.1 ± 1.93 13.8 ± 1.42 Flower, lavenderb
Terpine-4-ol 6.2 ± 1.00 8.2 ± 0.93 7.4 ± 0.99 9.5 ± 0.33 Pine-tree-likec,d
ρ-Cymene-8-ol 1.4 ± 0.02 5.1 ± 0.32 2.3 ± 0.66 7.3 ± 0.54 Herbaceouse
Myrtenol 18.3 ± 0.93 48.8 ± 5.20 41.9 ± 6.04 55.8 ± 4.20 Woody, mintyf
Verbenone 0.6 ± 0.27 3.1 ± 0.15 1.3 ± 0.31 3.8 ± 0.51 Minty camphoraceouse
Citronellol 14.0 ± 2.34 14.3 ± 1.28 13.4 ± 2.57 15.3 ± 3.42 Green lemonb
Geraniol 2.8 ± 0.16 4.0 ± 0.48 3.4 ± 0.77 5.0 ± 0.65 Floral, rose-likeb
∑ Terpenes 60.1 ± 5.91 103.1 ± 10.15 86.9 ± 13.77 119 ± 11.5
Esters Ethyl acetate 14.0 ± 0.16 15.6 ± 0.15 15.4 ± 0.17 16.1 ± 0.23 Solvent, fruityg
Ethyl lactate 10.2 ± 0.01 10.4 ± 0.06 10.7 ± 0.04 10.8 ± 0.06 Fruity, butteryh
Ethyl benzoate 49.3 ± 4.29 433.0 ± 8.63 357.1 ± 15.10 513.0 ± 7.53 Floral, fruitya
2-Phenylethyl acetate 2.5 ± 0.27 2.7 ± 0.06 2.6 ± 0.14 3.4 ± 0.57 Apple, roseb
∑ Esters 76 ± 4.73 461.7 ± 8.90 385.8 ± 15.45 543.3 ± 8.39
Alcohols 1-Propanol 123.8 ± 0.21 121.0 ± 0.38 124.0 ± 0.27 122.0 ± 0.47 Alcoholb
2-Methyl-1-propanol 259.9 ± 0.68 315.8 ± 0.97 321.0 ± 0.54 318.0 ± 0.48 Alcohol,banana.solventb
1-Butanol 3.7 ± 0.02 3.7 ± 0.02 3.4 ± 0.04 3.8 ± 0.01 Alcohol, fuselb
3-Methyl-1-butanol 1377.9 ± 2.87 1367.5 ± 4.33 1375.0 ± 5.21 1368.0 ± 2.46 Malty. fusela
2-Phenyl ethanol 38.4 ± 0.35 50.9 ± 0.12 46.3 ± 0.32 59.3 ± 0.45 Rose, sweetishi
Data are presented as means ± standard deviation (n = 3). Different letters indicate significant differences at p < 0.05 level
a
Pino et al. (18).
b
Vilanova et al. (19).
c
Noguerol-Pato et al. (20).
d
Choi (21).
e
Hognadottir et al. (22).
f
Nykanen and Suomalainen (23).
g
Duarte et al. (25).
h
Santiago and Rafael (26).
i
Liang et al. (27).

L 1) with AROMASE™ H2 was much higher than those of the
control wine (60.1 mg L 1), SUMIZYME AC (86.9 mg L 1) and
CYTOLASE PCL5 (103.1 mg L 1). The esters in KBR wine detected
by GC-FID were ethyl acetate, ethyl lactate, ethyl benzoate and
2-phenylethyl acetate. Ethyl esters are one of the most important
groups of aromatic compounds in KBR wine owing to their low
odour threshold (17,19,25,26). Ethyl acetate (solvent, fruity), ethyl
lactate (fruity, buttery) and 2-phenylethyl acetate (apple, rose)
experienced a slight increase in KBR wines treated with the three
commercial enzymes compared with the control wine. Among
the ester compounds, ethyl benzoate exhibited the highest rate
of increase following enzymatic treatment. The level of ethyl
benzoate was particularly high in the treated wines, attributed to
an ester bond between the benzene derivatives formed by the
enzyme and ethyl alcohol. The total ester content (543.3 mg L 1)
of AROMASE™ H2 was much higher than those of the control wine
(76.0 mg L 1), SUMIZYME AC (385.8 mg L 1) and CYTOLASE PCL5
(461.7 mg L 1). The alcohols in KBR wine detected via GC-FID were
1-propanol, 2-methyl-1-propanol, 1-butanol, 3-methyl-1-butanol
and 2-phenyl ethanol. Among these compounds, 3-methyl-1-
butanol was present in the highest concentration. The levels of
higher alcohols such as 1-propanol (alcohol), 2-methyl-1-propanol
(alcohol, banana, solvent), 1-butanol (alcohol, fusel) and 3-methyl-
1-butanol (malty, fusel) in KBR wine increased slightly or were
unaffected by the enzymatic treatment. 2-Phenyl ethanol
possesses a characteristic rose-like odour and a sweetish taste
(27). The concentration of 2-phenyl ethanol in AROMASE™ H2
(59.3 mg L 1) was higher than that of wine treated with CYTOLASE
PCL5 (50.9 mg L 1) or SUMIZYME AC (46.3 mg L 1). In view of
these results, the levels of terpenes, ethyl benzoate and 2-phenyl
ethanol in KBR wines treated with commercial enzymes increased
dramatically compared with the control wine, similar to the results
of previous reports (6–16). Terpenes, ethyl benzoate and 2-phenyl
ethanol impart a positive sensory character to KBR wine because
these compounds contribute to the floral and fruity aromas of
KBR wine. Commercial enzymes containing β-D-glucosidase
activity increased the concentrations of these compounds, helping
to increase the production of high-quality KBR wines on a
large scale. To enhance these compounds in KBR wine, three
commercial enzymes were added at the end of the alcohol
fermentation process. As a result of the enzymatic treatment
process, the concentrations of these compounds in all enzyme-
treated wines were higher than those of the control wine
(without-enzyme treatment). AROMASE™ H2 yielded the highest
quantity of these compounds among the three commercial
enzymes. A histogram of the total aromatic concentration in KBR
wines treated with three different commercial enzymes is shown
in Fig. 1. The order of total aromatic abundance was as follows:
AROMASE™ H2 > CYTOLASE PCL5 > SUMIZYME AC. The highest
total aromatic concentration was found in KBR wine treated with

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Volatiles in black raspberry wine via enzymatic treatment
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Figure 1. Histogram of aromas in Korean black raspberry (KBR) wines treated with different commercial enzymes. Figure is reproduced in colour in online version. [Colour figure
can be viewed at wileyonlinelibrary.com]

AROMASE™ H2 (∑ Aroma =721.6), whereas the lowest total
aromatic concentration was found in KBR wine treated with
SUMIZYME AC (∑ Aroma =519.0).
PCA of active aromatic compounds
PCA was applied to the relationships between the various enzymes
and the concentration of terpenes, esters and 2-phenyl ethanol
from Table 1. The first principal component (PC1) accounted for
87.5% of the total variance, whereas the second principal
component (PC2) accounted for an additional 9.2% of the total
variance (Fig. 2). KBR wine treated with AROMASE™ H2 was
positioned in the upper right quadrant, owing to the presence of
ρ-cymene, β-linalool, ρ-cymene-8-ol, verbenone, citronellol,
geraniol and phenylethyl acetate. These compounds possess
aromatic description such as ‘citrus’, ‘flower’, ‘minty’, ‘lemon’, ‘floral’
and ‘rose’ (18,19,22).
In the lower right quadrant, KBR wine treated with CYTOLSE
PCL5 was placed primarily owing to the presence of terpine-4-ol,
myrtenol, ethyl acetate, ethyl lactate, ethyl benzoate and 2-phenyl
ethanol, which possess agreeable aroma descriptions, such
as ‘pine-tree like’, ‘minty’, ‘fruity’, ‘butter’, ‘floral’ and ‘rose’
(18,20,21,23,25–27).
KBR wine treated with SUMIZYME AC (lower left quadrant) was
slightly off with regard to the active aromatic compounds
(terpenes, esters and 2-phenyl ethanol). The control wine (upper
left quadrant) was far from the active aromatic compounds. The
distribution of samples in the consensus space was indicative of
the differences among the wines treated with three commercial
enzymes and the control wine. As shown in Fig. 2, the aromatic
characters of the wines treated with AROMASE™ H2 and CYTOLASE

Figure 2. Principal component analysis (PCA) of active aroma compounds in KBR wines treated with different commercial enzymes. 1, Control wine; 2, CYTOLASE PCL5; 3,
SUMIZYME AC; 4, AROMASE™ H2. [Colour figure can be viewed at wileyonlinelibrary.com]

J. Inst. Brew. 2017 Copyright © 2017 The Institute of Brewing & Distilling wileyonlinelibrary.com/journal/jib

Institute of Brewing & Distilling
Figure 3. Quantitative descriptive of analysis of KBR wines treated with different commercial enzymes. Figure is reproduced in colour in online version. [Colour figure can be
viewed at wileyonlinelibrary.com]
PCL5 were distinctly different from that of wine treated with
SUMIZYME AC and the control wine.
Sensory evaluation for preference
Sensory evaluations of the enzyme-treated KBR wines and control
wine were performed by 25 trained panellists based on a
quantitative descriptive analysis. ANOVA was used to differentiate
the enzyme-treated wines through evaluation scores. As shown in
Fig. 3, wine treated with CYTOLASE PCL5 achieved the highest
values for fruity and floral aromas. Wine treated with SUMIZYME
AC achieved higher assessment scores than the control wine. In
contrast, wine treated with AROMASE™ H2 achieved the lowest
scores for overall acceptability owing to musty and earthy off-
odours, although it possessed the highest total quantity of
aromatic compounds. AROMASE™ H2 is a commercial enzyme
prepared from Penicillium multicoloris. The musty and earthy
off-odours could be attributed to geosmin produced from the
metabolism of Penicillium spp. (28). Geosmin was expected to have
the greatest influence on the off-odour of wine owing to its
extremely low odour threshold (50 ng L 1) (29). The order of total
acceptability in terms of aromatic preference was wine treated
with CYTOLASE PCL5 (4.9) > SUMIZYME AC (4.1) > control wine
(3.9) > AROMASE™ H2 (3.1) (Fig. 3).
Conclusions
In our previous study, we selected the optimal yeast strain for the
production of KBR wine with good acceptability. In order to further
improve the flavour quality of KBR wine fermented with the
selected yeast strain on a large scale, it was important for us to
identify the best commercial enzyme that can increase the
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B.-H. Kim and S. K. Park
concentration of aromatic compounds associated with floral
(terpenes) and fruity (esters) characters. In this study, KBR wines
treated with CYTOLASE PCL5, SUMIZYME AC and AROMASE™ H2
exhibited higher concentrations of aromatic compounds than
the control wine. Among the commercial enzymes, KBR wine
treated with CYTOLASE PCL5 showed high concentration of
aromatic compounds and highest scores of fruity and floral aromas
by sensory evaluation. This study would help to produce high-
flavour quality KBR wines in large-scale production.
Acknowledgements
DSM Food Specialities is thanked for supplying and preparing the
commercial enzymes and for technical advice. We would also like
to thank Jin-kook Kim, the director of HiteJinro R&D, who allowed
this study and made its publication possible. There are no conflicts
of interest to declare.
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