Duarte et al., 2020 - Differentiation of aromatic, bittering and dual-purpose commercial hops from their

Food Research International 138 (2020) 109768
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Food Research International
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Differentiation of aromatic, bittering and dual-purpose commercial hops
from their terpenic profiles: An approach involving batch extraction,
GC–MS and multivariate analysis
Lucas Mattos Duarte a,b , Tatiane Lima Amorim a , Richard Michael Grazul c , Marcone Augusto
Leal de Oliveira a,*
a Grupo de Química Analítica e Quimiometria (GQAQ), Departamento de Química, Universidade Federal de Juiz de Fora, 36036900 Juiz de Fora, MG, Brazil
b Departamento de Química Analítica, Instituto de Química, Universidade Federal Fluminense, 24020-141 Niterói, RJ, Brazil
c Laboratório de Pesquisa em Química Medicinal e Produtos Naturais, Departamento de Química, Universidade Federal de Juiz de Fora, 36036900 Juiz de Fora, MG,
Brazil
ARTICLE INFO
Keywords:
Hops
Humulus lupulus
Terpenes
Aroma
Principal component analysis
Beer
ABSTRACT
Terpenes are one of the main classes of compounds in hops (Humulus lupulus, L). They play an important role in
brewing due to their central function, which is related to beer aroma. A screening of terpenes in several commercial
hop varieties was carried out by gas chromatography coupled to mass spectrometry after employing a
simple, straightforward and high throughput extraction method. A single batch extraction, using hexane as
solvent, was employed to obtain the terpenic fraction of the hop samples. Nineteen terpenes were identified in
analyzed samples with β-myrcene (2.22–45.30%), α-humulene (20.20–67.64%), and β-caryophyllene
(9.97–24.62%) being the major terpenes in all samples. The studied system was multivariate modeled by principal
component analysis. Based on the proposed approach, it was possible to correlate the terpenic hop profiles
to their specific purpose in the brewing industry and to distinguish aromatic hops (high α-humulene content),
bittering hops (high β-myrcene content), and dual-purpose hops (more complex and intermediate terpenic
profile) among the samples.
1. Introduction
Hops are the most complex and expensive raw material used in the
brewing industry. The volatile components of hops (the essential oil) are
a complex mixture comprising hundreds of compounds, which are made
up largely of terpenes (~70%) (Almaguer, Schönberger, Gastl, Arendt, &
Becker, 2014). Dry hops contain 0.5–3.0% of essential oil (Eyres, Marriott,
& Dufour, 2007). The main terpenes encountered are the monoterpene
β-myrcene and the sesquiterpenes β-caryophyllene and
α-humulene (Inui, Tsuchiya, Ishimaru, Oka, & Komura, 2013). These
compounds account for up to 80% of the total volatiles of hop cultivars
bred for the brewing industry (Rettberg, Biendl, & Garbe, 2018).
Moreover, some hop varieties present significant amounts of different
terpenes, such as farnesene, salinene, and bergamotene (Bernotienë,
Nivinskienë, Butkienë, & Mockutë, 2004; Steinhaus & Schieberle, 2000).
Although terpenes are the major constituents of the hop essential oil,
many other substances are found in this matrix, including esters, aldehydes,
ketones, and alcohols (Gonçalves, Figueira, Rodrigues, &
Câmara, 2012; Sharpe & Laws, 1981).
The hop essential oil is extracted along with α-acids – precursors of
beer bitterness – in the wort boiling step, and the occurrence of bitter or
aromatic characteristics in the final product depends on several factors.
These include the chemical composition, the time of hop addition, as
well as the hop amount added (Štěrba et al., 2015). After hop addition,
part of their volatiles evaporate, and other processes may occur, such as
thermal conversion and oxidation (Haley & Peppard, 1983). In the final
product, only trace levels of monoterpenes and sesquiterpenes are
found. However, significant concentrations of polar oxygenated terpene
derivatives such as humulene epoxides and linalool oxides, ethers, ketones,
and esters are found, which contribute to the desired flavor and
aroma of beers (Tressl, Friese, Fendesack, & Koeppler, 1978). The
quality improvement of beer aroma may be achieved by adding different
* Corresponding author at: Departamento de Química da Universidade Federal de Juiz de Fora (Rua José Lourenço Kelmer – São Pedro), 36036900 Juiz de Fora,
MG, Brazil.
E-mail address: marcone.oliveira@ufjf.edu.br (M.A.L. de Oliveira).
https://doi.org/10.1016/j.foodres.2020.109768
Received 12 May 2020; Received in revised form 8 September 2020; Accepted 29 September 2020
Available online 6 October 2020
0963-9969/© 2020 Elsevier Ltd. All rights reserved.

L.M. Duarte et al.
hop varieties, performing the late addition of hops, or adding a pure
aroma hop extract to create different hoppy flavors (Oladokun et al.,
2017; Vollmer & Shellhammer, 2016).
Gas chromatography coupled to mass spectrometry (GC–MS) is an
extremely powerful technique in hop research to characterize the
terpene fraction. It is undoubtedly the method of choice for the analysis
of minor volatiles from hops and beers (Bernotienë et al., 2004; Ceola,
Huelsmann, Da-Col, & Martendal, 2019; Forteschi et al., 2019; Gonçalves
et al., 2012; Kishimoto, Wanikawa, Kagami, & Kawatsura, 2005;
Leonardi et al., 2013; Mongelli et al., 2016; Praet et al., 2015; Van
Opstaele, De Causmaecker, Aerts, & De Cooman, 2012). The technique
permits the separation of over 200 compounds in a single chromatographic
run (Ligor et al., 2014) , and the mass spectral data greatly facilitates
constituent identification with high accuracy, being also able to
detect peak overlapping. GC with flame ionization detection (FID) is also
very useful for terpenes studies in hops, mainly considering a quantitative
approach (Kenny et al., 2004; Killeen et al., 2017; Rettberg et al.,
2018). Some reference methods are available considering GC-FID for the
quantification of terpenes in hops essential oil (American Society of
Brewing Chemists, 2016b; European Brewing Convention, 2006).
With respect to sample preparation, hydrodistillation is the most
commonly employed extraction method to obtain the hop essential oil.
This process requires a considerable amount of the plant, and total
extraction time may take more than 4 h per sample (Shellie et al., 2009).
The reference method by steam distillation also reports the use of a
considerable amount of sample (more than 100 g) (American Society of
Brewing Chemists, 2016a). However, less sample mass can be used in
adapted steam distillation systems (De Cooman, Everaert, & De Keukeleire,
1998). Other methods have also been employed, such as direct
extraction with solvents (Steinhaus & Schieberle, 2000), supercritical
fluid extraction (Van Opstaele, Goiris, De Rouck, Aerts, & De Cooman,
2012; Zekovic, Pfaf-Sovljanski, & Grujic, 2007), and solid-phase
extraction using stir bar-sorption (Kishimoto et al., 2005; Steyer,
Clayeux, & Laugel, 2013). Headspace analysis, directly coupled to GC
(Ceola et al., 2019; Gonçalves et al., 2012; Jorge & Trugo, 2003; Van
Opstaele, De Causmaecker, Aerts, & De Cooman, 2012) was also used for
the analysis of hop volatiles.
This work aimed to perform a terpenic screening in 25 different
commercial hop varieties and evaluate the possibility of distinguishing
them in accordance with their terpenic profile employing multivariate
analysis. The terpenes fraction was obtained after a simple, fast, and
straightforward extraction method, considering a batch protocol to
prepare samples simultaneously. The characterization of volatiles was
carried out by GC–MS. The sample pattern recognition was performed
by using the terpenes common to all hop samples, obtained by GC–MS,
and principal component analysis (PCA).
2. Materials and methods
2.1. Samples and reagents
Twenty-five commercial hop samples from different varieties were
acquired at a Brazilian market (Juiz de Fora/MG) within the period of
July-August/2019. These hop varieties along with their labeled α-acid
content were: 1) Amarillo (12.6%), 2) Fuggle US (5.0%), 3) Target
(9.0%), 4) Saaz US (3.5%), 5) Cascade (6.1%), 6) Amarillo (5.5%), 7)
Mosaic (12.0%), 8) Mittelfruh (4.8%), 9) El dorado (10.5%), 10) Er
golding (5.4%), 11) Northern Brewer (10.4%), 12) Hersbrucker (2.5%),
13) Magnum (14.9%), 14) Columbus (16.3%), 15) Citra (13.7%), 16)
Nugget (12.0%), 17) Columbus (14.9%), 18) Herkules (14.3%), 19)
Simcoe (12.0%), 20) Saaz (3.0%), 21) Tradition (3.0%), 22) S. Golding
(4.0%), 23) Perle (4.2%), 24) Fuggle (4.0%), 25) Tettnang (2.2%). Dried
hops in pellets, marketed and sealed under vacuum, were used for all
experiments. Samples were stored under − 20 ◦ C until their use.
All reagents and solvents used were of analytical grade. Hexane
(HEX) and sodium sulfate were acquired from Sigma-Aldrich (St. Louis,
Food Research International 138 (2020) 109768
MO, EUA). Dichloromethane (DCM) was purchased from Vetec (Rio de
Janeiro, RJ, Brazil). A standard solution, containing C8-C20 linear alkanes,
was acquired from Sigma-Aldrich (St. Louis, MO, EUA).
2.2. Extraction procedures
2.2.1. Hydrodistillation (HD)
The hop sample in pellets was pulverized using an agate mortar and
pestle. 1 g of the sample was weighed and transferred into a 100 mL
round bottom extraction flask, to which 50 mL of deionized water was
added. A 45 ◦ glass adapter was used to attach the round bottom to the
Liebig condenser, and a 25 mL graduated test tube was employed at the
outlet of the condenser to collect approximately 20 mL of the hydrodistillate.
A heating mantle with a magnetic stirrer was used to heat and
homogenize the solution, and the temperature was kept between 120
and 130 ◦ C. After hydrodistillation, liquid–liquid extraction with HEX
was performed, and the solution was filtered through a glass pipette
filled with cotton and sodium sulfate (Na 2 SO 4 ). The solvent was evaporated
in a rotary film evaporator at 40 ◦ C under reduced pressure.
Subsequently, the essential oil was reconstituted in 1 mL of HEX and
transferred into a GC vial to be analyzed by GC–MS immediately.
2.2.2. Ultrasound-assisted solvent extraction (UASE)
This extraction procedure was evaluated employing HEX and DCM
solvents. 200 mg of pulverized hop sample was transferred into a glass
tube, to which 1 mL of solvent was added. The tube was closed with a
Teflon R cap and maintained in an ultrasound bath (220 Watts, 40 kHz)
during one hour at 25 ◦ C. The extract was filtered through a pipette
containing a cotton plug and Na 2 SO 4 into a GC vial and immediately
analyzed by GC–MS.
2.3. GC–MS instrumentation and peaks identification
Terpene analysis was performed on a Shimadzu QP2010Plus GC
equipped with an electron impact ionization source, a single quadrupole
mass analyzer, an AOC-5000 auto-sampler and a Restek Rtx-5MS column
(5% diphenyl/95% dimethyl-polysiloxane, 30 m × 0.25 mm i.d.,
0.25 µm d f ). The temperature program used was as follows: initial oven
temperature, 50 ◦ C; after 5 min, the oven temperature was raised to
200 ◦ C at the rate of 4 ◦ C min − 1 and then held for 5 min. The total run
time was 47.5 min. An injection volume of 1.0 µL and a split ratio of 1:10
were used. Injector, ion source, and interface temperatures were 200 ◦ C,
200 ◦ C, and 220 ◦ C, respectively. Helium was used as carrier gas at a
flow of 1.0 mL min − 1 . Mass fragmentation patterns were produced at 70
eV ionization energy, and mass to charge ratio scanned was from 40 to
700 m/z. The solvent cut-off time was 5 min. Area normalization was
used for expressing terpenes relative contents in samples.
Peak identification was carried out considering three parameters as
follows: 1) evaluating the mass spectrum of the target peak with that
suggested by NIST08 and NIST08s libraries, taking into account higher
similarity percentages; 2) comparing the calculated retention index (RI)
with the RI values available in the literature (Adams, 2007); 3) checking
the elution order of the analytes with studies described in the literature
considering the same stationary phase (Gonçalves et al., 2012; Ligor
et al., 2014; Mongelli et al., 2016).
RI was calculated based on Van den Dool and Kratz (Van Den Dool &
Kratz, 1963) (Eq. (1)), where, tr i is the analyte retention time (target
terpene) in the sample chromatogram, tr n and tn+1 r are the retention times
of the alkanes eluting immediately before and after the target peak,
respectively, and n is the carbon number of the alkane that elutes
immediately before the analyte:
( ) t
i
RI = 100n + r
− tr
n × 100 (1)
tr
n+1 − tr
n
2

L.M. Duarte et al.
2.4. Statistical analysis
The multivariate analysis was performed by employing PCA. The
relative contents of terpenes identified in all 25 hop samples after UASE
extraction with hexane were considered in the input matrix. It is
Food Research International 138 (2020) 109768
important to highlight that although a greater number of terpenes were
identified (Table 1), only 13 terpenes found in all hop samples were used
to perform PCA: A) β-myrcene, B) linalool, C) geraniol, D) α-copaene, E)
β-caryophyllene, F) β-cubebene, G) α-humulene, H) γ-muurolene, I)
β-selinene, J) α-selinene, K) α-muurolene, L) γ-cadinene and M)
Table 1
Comparison among extraction methods: hydrodistillation (HD) and ultrasound-assisted solvent extraction (UASE) with hexane (HEX) and dichloromethane (DCM),
considering retention time (t r ) of analytes (min), relative content (%), calculated retention index (RI calc ) , literature retention index (RI lit ), and similarity between the
target mass spectrum and NIST library (%).
Peak Identification MF Extraction t r Content RI calc RI lit Similarity
1 β-myrcene C 10 H 16 HD 12.05 28.68 994 988 96
UASE-HEX 12.06 25.05 995 95
UASE-DCM 12.06 46.90 994 97
2 limonene C 10 H 16 HD 13.63 0.44 1032 1024 80
UASE-HEX 13.63 0.78 1032 90
UASE-DCM 13.63 0.72 1032 82
3 linalool C 10 H 18 O HD 16.67 1.57 1104 1095 90
UASE-HEX 16.66 0.84 1104 97
UASE-DCM 16.68 0.58 1105 76
4 geraniol C 10 H 18 O HD 22.77 1.24 1262 1249 90
UASE-HEX 22.74 2.10 1261 96
UASE-DCM n.d. – n.d. –
5 α-copaene C 15 H 24 HD 27.12 0.34 1385 1374 73
UASE-HEX 27.12 0.80 1385 93
UASE-DCM 27.13 0.25 1385 67
6 β-caryophyllene C 15 H 24 HD 28.64 14.45 1431 1417 95
UASE-HEX 28.65 15.22 1431 96
UASE-DCM 28.64 10.91 1431 95
7 β-cubebene C 15 H 24 HD 28.95 0.08 1440 1387 –
UASE-HEX 28.95 0.34 1440 90
UASE-DCM n.d. –. n.d. –
8 α-bergamotene C 15 H 24 HD 29.10 0.65 1445 1432 83
UASE-HEX 29.11 1.07 1445 95
UASE-DCM 29.11 0.37 1445 76
9 α-humulene C 15 H 24 HD 29.79 42.20 1466 1452 95
UASE-HEX 29.81 41.41 1467 95
UASE-DCM 29.78 36.33 1466 95
10 γ-muurolene C 15 H 24 HD 30.51 1.51 1488 1478 89
UASE-HEX 30.50 2.26 1488 92
UASE-DCM 30.50 0.73 1488 –
11 β-selinene C 15 H 24 HD 30.87 2.06 1499 1489 93
UASE-HEX 30.87 2.14 1499 93
UASE-DCM 30.87 0.84 1499 75
12 α-selinene C 15 H 24 HD 31.15 2.09 1508 1498 90
UASE-HEX 31.15 2.63 1508 91
UASE-DCM 31.15 1.00 1508 –
13 α-muurolene C 15 H 24 HD 31.27 0.21 1512 1500 75
UASE-HEX 31.27 0.12 1512 90
UASE-DCM n.d. – n.d. –
14 α-farnesene C 15 H 24 HD 31.41 0.24 1516 1505 80
UASE-HEX 31.39 0.38 1516 93
UASE-DCM n.d. – n.d. –
15 γ-cadinene C 15 H 24 HD 31.73 1.46 1527 1513 90
UASE-HEX 31.73 1.75 1527 90
UASE-DCM 31.74 – 1527 75
16 δ-cadinene C 15 H 24 HD 32.00 2.56 1536 1522 89
UASE-HEX 32.00 2.68 1536 89
UASE-DCM 32.00 – 1536 75
17 eremophilene C 15 H 24 HD 32.31 0.11 1546 1512 –
UASE-HEX 32.30 0.2 1546 91
UASE-DCM n.d. – n.d. –
18 eudesma-3,7(11)-diene C 15 H 24 HD 32.47 0.11 1551 1535–1542 –
UASE-HEX 32.46 0.22 1551 91
UASE-DCM n.d. – n.d. –
n.d. – not detected | MF – molecular formula.
3

L.M. Duarte et al.
δ-cadinene. Then, a data matrix of 25x13 was used (25 samples in lines
and 13 terpenes as variables in columns). Data were mean-centered
before the multivariate modeling, and PCA was based on covariance
using 3 principal components (PC). All calculations were performed on
Origin 2016.
The statistical comparison between the most promising extraction
methods (HD and UASE with HEX) was carried out using the nonparametric
Wilcoxon rank-sum test, since normality assumption was
violated. Data normality was investigated by applying the Ryan-Joyner
test (within 95% of confidence) in the residues of terpenes calculated
content when using HD and UASE with HEX. Both statistical tests were
carried out on Minitab 14 software.
3. Results and discussion
3.1. Comparison among extraction methods
Fig. 1 shows chromatograms for the comparison of three different
extraction procedures to access the terpenic profile of hops: HD
(Fig. 1A), UASE with HEX (Fig. 1B), and UASE with DCM (Fig. 1C).
Columbus variety was employed for this preliminary study. Chromatograms
depicted in Fig. 1A and B allowed to identify 18 terpenes: 1)
β-myrcene, 2) limonene, 3) linalool, 4) geraniol, 5) α-copaene, 6)
β-caryophyllene, 7) β-cubebene, 8) α-bergamotene, 9) α-humulene, 10)
γ-muurolene, 11) β-selinene, 12) α-selinene, 13) α-muurolene, 14)
α-farnesene, 15) γ-cadinene, 16) δ-cadinene, 17) eremophilene and 18)
eudesma-3,7(11)-diene. Peaks 14, 17, and 18 were not observed in the
chromatogram of Fig. 1C. All mono and sesquiterpenes eluted within 35
min.
Retention time (t r ) of the analytes, relative terpene content, calculated
retention index (RI calc ) , the retention index available in the literature
(RI lit ) (Adams, 2007), and similarity between the target mass
spectrum and the NIST libraries are reported in Table 1.
The main terpenes β-myrcene, β-caryophyllene, and α-humulene
represented the major peaks, as can be seen in the chromatograms of the
Food Research International 138 (2020) 109768
three extraction approaches (Fig. 1). However, their relative contents
were different when comparing HD, UASE with HEX, and UASE with
DCM (Table 1) due to the type of extraction solvent. The sesquiterpenes
β-caryophyllene and α-humulene present similar structures and are
more non-polar than the monoterpene β-myrcene. Thus, sesquiterpenes
were better extracted with hexane, a non-polar solvent. On the other
hand, the extraction with DCM, a more polar and aprotic solvent,
favored the extraction of the monoterpene β-myrcene.
HD and UASE with HEX led to similar contents of major terpenes:
28.68% and 25.05% (β-myrcene), 14.45% and 15.22% (β-caryophyllene);
42.20% and 41.42% (α-humulene), respectively. On the
other hand, UASE with DCM led to lower contents of β-caryophyllene
and α-humulene (10.91% and 36.33%) and a higher content of β-myrcene
(46.90%), when compared to the others two approaches. Additionally,
using UASE with DCM, peaks related to geraniol, β-cubebene,
α-muurolene, α-farnesene, eremophilene, and eudesma-3,7(11)-diene
were not identified due to the poor extraction of these compounds
when DCM was employed. Furthermore, γ-muurolene and α-selinene,
previously identified by the other extraction approaches, could not be
assigned with confidence due to the low similarity provided by the library
(see Table 1).
In addition, HD and UASE with HEX procedures were statistically
compared. The nonparametric Wilcoxon rank-sum test was performed
for this comparison since normality was not verified (p-value < 0.01).
The 95% confidence interval was considered in both tests. The
nonparametric test resulted in a p-value of 0.122, indicating no significant
difference between both extraction approaches
In this regard, UASE with HEX was defined as the extraction procedure
to be employed to access the terpenic fraction of all commercial
hop varieties. This proposed extraction approach was simpler than HD
(the classical approach) and provided similar extraction of terpenes
(Table 1). Furthermore, only 200 mg of sample and 1 mL of HEX were
necessary to perform the extraction. All 25 commercial hop samples
were simultaneously prepared in a single batch, in just 1 h.
Table 2 shows the terpenic profile of 25 different hop extracts. As
Fig. 1. Chromatograms of hop extracts obtained by hydrodistillation (A) ultrasound-assisted solvent extraction with hexane (B) and ultrasound-assisted solvent
extraction with dichloromethane (C). The dashed rectangle represents an expansion of the region from 28 to 33 min for all approaches. Identified terpenes in sample:
1: β-myrcene; 2: limonene; 3: linalool; 4: geraniol; 5: α-copaene; 6: β-caryophyllene; 7: β-cubebene; 8: α-bergamotene; 9: α-humulene; 10: γ-muurolene; 11: β-selinene;
12: α-selinene; 13: α-muurolene; 14: α-farnesene; 15: γ-cadinene; 16: δ-cadinene; 17: eremophilene; 18: eudesma-3,7(11)-diene.
4

L.M. Duarte et al.
Table 2
Terpene composition of different commercial hop varieties analyzed after an ultrasound-assisted solvent extraction with hexane. Relative content expressed in percentage.
β-myrcene limonene linalool geraniol α-copaene β-
β-cubebene α5- α-humulene γ-muurolene β-selinene α-selinene α- α-farnesene γ-cadinene δ-cadinene eremophilene eudesmacaryophyllene
bergamotene
muurolene
3,7(11)-
diene
alloaroma
dendrene
5
Amarillo (1) 39.50 0.98 0.77 0.62 0.70 17.20 0.46 n.d. 25.90 2.22 3.26 3.73 0.22 n.d. 1.44 2.30 0.45 0.34 n.d.
Fuggle (2) 10.80 0.82 3.54 0.55 0.54 9.97 0.32 1.97 36.90 2.71 5.64 6.60 0.48 0.29 1.28 2.99 6.22 5.94 2.48
Target (3) 5.50 0.59 1.90 0.52 2.00 17.30 1.32 N.d. 36.60 5.41 2.91 2.25 1.47 n.d. 5.83 8.01 3.14 5.26 n.d.
Saaz (4) 13.10 0.57 1.28 0.47 0.45 11.70 0.38 2.15 62.70 1.35 0.30 0.44 0.26 0.20 1.68 2.53 0.21 0.24 n.d.
Cascade (5) 21.80 0.49 0.73 2.41 0.75 13.60 0.31 0.91 43.20 2.08 3.82 4.27 0.36 0.53 1.68 2.62 0.29 0.15 n.d.
Amarillo (6) 23.30 0.46 1.35 1.06 0.62 14.90 0.58 0.98 47.30 1.73 0.43 0.44 0.59 0.48 2.05 3.28 0.34 0.11 n.d.
Mozaic (7) 45.30 1.33 1.34 2.19 0.65 13.70 0.61 n.d. 27.40 1.30 0.78 0.26 0.26 0.52 1.48 2.41 0.30 0.26 n.d.
Mittelfruh 11.20 0.42 1.55 0.22 0.88 19.00 1.00 n.d. 49.10 2.49 1.45 1.59 0.83 0.19 2.96 4.50 1.24 1.04 0.36
(8)
El dorado (9) 31.60 0.58 1.11 2.16 1.08 17.40 0.97 n.d. 22.50 3.02 4.03 4.60 0.74 0.34 2.83 4.50 1.49 1.07 n.d.
Er Golding 14.10 0.42 0.83 0.20 0.80 20.20 1.15 n.d. 51.60 2.02 0.80 0.65 0.48 0.11 2.37 3.67 0.44 0.19 n.d.
(10)
Northern 27.90 0.94 0.74 0.27 0.98 19.00 0.97 n.d. 37.80 2.07 0.88 0.41 0.64 0.18 2.49 3.97 0.45 0.40 n.d.
Brewer
(11)
Hersbrucker 4.86 n.d. 1 1.09 0.14 0.55 15.30 0.70 n.d. 36.50 2.92 6.98 7.93 0.37 n.d. 1.43 3.30 7.53 6.24 4.22
(12)
Magnum 29.00 0.85 0.76 0.53 0.73 17.10 1.26 n.d. 39.20 1.79 0.77 0.79 0.29 1.18 1.86 3.22 0.40 0.35 n.d.
(13)
Columbus 29.80 0.79 0.99 3.40 1.87 15.60 1.16 n.d. 21.10 3.78 2.47 2.90 1.03 n.d. 3.83 5.77 3.04 2.52 n.d.
(14)
Citra (15) 44.30 0.95 2.09 0.68 0.48 15.80 0.39 n.d. 22.70 1.27 3.08 2.81 0.22 1.41 1.27 2.06 0.19 0.24 n.d.
Nugget (16) 33.80 0.88 1.90 0.16 0.79 18.20 0.63 n.d. 31.00 1.73 2.65 2.49 0.33 0.33 1.69 2.78 0.37 0.37 n.d.
Columbus 31.70 0.75 1.10 2.56 1.50 15.40 1.75 n.d. 20.20 3.77 2.69 2.65 1.20 n.d. 3.92 5.77 2.94 2.18 n.d.
(17)
Herkules 21.60 1.19 0.71 0.71 1.22 17.50 0.74 n.d. 43.60 1.97 1.07 0.95 0.63 0.23 2.48 3.99 0.80 0.70 n.d.
(18)
Simcoe (19) 34.20 1.24 1.40 1.97 1.20 16.90 0.45 n.d. 31.50 1.98 0.88 0.45 0.46 0.46 2.26 3.61 0.48 0.58 n.d.
Saaz (20) 5.16 0.25 1.06 0.57 0.49 13.23 0.52 2.02 67.64 1.31 0.33 0.27 0.28 n.d. 1.69 2.88 0.55 1.75 n.d.
Tradition 5.05 0.36 2.20 0.14 0.74 20.95 0.94 n.d. 58.68 1.96 0.78 0.46 0.54 n.d. 2.51 3.90 0.39 0.42 n.d.
(21)
S. Golding 2.22 0.10 2.21 0.41 1.06 18.01 0.71 0.92 39.23 4.08 11.89 10.67 0.66 n.d. 2.97 4.05 0.56 0.25 n.d.
(22)
Perle (23) 6.89 0.29 0.51 0.18 0.85 24.62 1.19 n.d. 53.68 1.95 0.91 0.42 0.67 n.d. 2.49 4.35 0.65 0.37 n.d.
Fuggle (24) 3.78 1.01 4.78 1.14 0.76 11.07 0.38 2.45 31.69 3.82 8.24 7.68 0.42 n.d. 1.90 3.76 7.06 6.04 4.02
Tettnang
(25)
13.14 0.43 1.16 0.42 0.36 11.99 0.56 1.83 60.06 1.32 0.93 0.98 0.31 n.d. 1.51 2.66 0.66 1.69 n.d.
n.d. – not detected.
Food Research International 138 (2020) 109768

L.M. Duarte et al.
expected, the major compounds for all samples were β-myrcene, β-caryophyllene,
and α-humulene. Linalool, geraniol, α-copaene, β-cubebene,
γ-muurolene, β-selinene, α-selinene, α-muurolene, γ-cadinene, and
δ-cadinene were identified in all samples, albeit in much lower relative
contents. Furthermore, α-bergamotene was found in 9 samples, α-farnesene
was found in 20 samples and alloaromadendrene, not previously
identified in the bittering Columbus hop (used to evaluate the extraction
procedure), was identified in some aromatic hops, such as Fuggle
(samples 2 and 24), Mittelfruh (sample 8) and Hersbrucker (sample 12).
3.2. Evaluation of hop samples based on their terpenic profile
A multivariate approach employing PCA was carried out in order to
recognize similar patterns within the studied set of hop samples. Thirteen
terpenes, present in all 25 samples, were used as variables. Fig. 2
shows the biplot scores graph of PCA (black axis) along with the loading
information for the first two PCs (blue axis). Four major groups could be
highlighted in Fig. 2 based on the terpenic profile of the hop samples.
PC1, responsible for 75.31% of the explained variance, clearly distinguished
hop groups based on β-myrcene (A) and α-humulene (G) content,
since these two terpenes were the most relevant variables for this
observed grouping. Hops with the highest β-myrcene content were
highlighted by the red ellipse, being grouped in the positive scores of
PC1 (right side). On the other hand, samples with the highest content of
α-humulene, were grouped in negative scores of PC1 (green and black
ellipses, respectively). PC2 was responsible for 19.32% of the explained
variance. This new latent variable played an important role in highlighting
other terpenes as significant original variables, and it was able
to differentiate hops with a more diversified terpenic profile. Hops with
intermediate characteristics were grouped close to the zero of PC1 and in
the positive scores of PC2 (orange ellipse). PC3 was responsible for
3.21% of the explained variance, and 98.24% of the total explained
variance was observed for the three PCs. PC1 × PC2 score plot was most
suitable to be shown since no substantial information on clustering was
found when PC3 was considered.
Fig. 3 shows the contents of the three major terpenes for the studied
Fig. 2. Biplot scores graph to hops grouping according to their terpenic profile
(black axis) and loading plot (blue axis). Red ellipse: hops with high β-myrcene
content (variable A), which also presented bittering features; Black ellipse: hops
with very high α-humulene content (variable G), which also presented aromatic
features; Green ellipse: hops with high α-humulene content and aromatic features,
but with the sum of the three major terpenes lower than 60%; Orange
ellipse: hops with an intermediate terpenic profile, where some dual-purpose
hops were identified (high α-humulene and high α-acid contents). (For interpretation
of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
Food Research International 138 (2020) 109768
samples, as well as the α-humulene/β-myrcene and α-humulene/β-caryophyllene
ratios. Both ratios are important in brewing and can be
related to bitter and aromatic properties of hops (Bernotienë et al.,
2004). High ratios usually characterize aromatic hops, while low ratios
usually characterize bitter hops. This figure was constructed to better
interpret hop grouping according to their terpenic profile and to investigate
some correlation between terpene profiles and α-acid labeled
content of commercial hops. Graphs were arranged following the four
groups depicted in the PCA (red ellipse corresponds to Fig. 3A, black
ellipse corresponds to Fig. 3B, green ellipse corresponds to Fig. 3C, and
orange ellipse corresponds to Fig. 3D).
In Fig. 3A, hop samples with the highest β-myrcene contents (greater
than 20%) can be observed. The ratio α-humulene/β-myrcene for this
group was very low, and values around 1.0 were observed for these
samples with higher β-myrcene content. Low ratios of α-humulene/
β-caryophyllene were also found (around 1.5). Literature reports that
β-myrcene production is characteristic of bitter hops (Leonardi et al.,
2013), which is in agreement with the results obtained in the present
study since samples with higher α-acid contents also presented higher
β-myrcene contents. Samples 9, 14, and 17 showed the lowest contents
of the three major terpenes (<80%) when compared to the other samples
in the same group, which explains why these samples were located in
negative scores of PC2, while all others were located in the positive
scores (Fig. 2).
Fig. 3B shows hop samples with the highest α-humulene content (up
to 50%). In these samples, the sum of the three major terpenes was
usually higher than 80%. Furthermore, some hops classified as the finest
aroma hops in the literature (Leonardi et al., 2013) could be identified
within this group (samples 4, 20, and 25, which are Saaz, Saaz, and
Tettnang, respectively). For these hops, the ratio α-humulene/β-caryophyllene
was around 5.0. Fig. 3C also shows samples with low
β-myrcene and high α-humulene content, which justified the grouping in
the negative scores of PC1 (Fig. 2). However, these samples had the
lowest content of α-humulene, β-myrcene, and β-caryophyllene (usually
less than 60%). It can be observed that samples within this group (green
ellipse in PCA) had the lowest α-acids content and also the most significant
ratios of humulene/β-myrcene and α-humulene/β-caryophyllene.
As such, the aromatic hops had a terpenic profile with low
β-myrcene and high α-humulene content, while the hops classified as the
finest aroma hops had the highest amounts of α-humulene among all
samples analyzed.
Fig. 3D shows the profile of the major terpenes for samples located
close to the zero of PC1 and in positive scores of PC2, grouped in the
orange ellipse. These samples contained a total of roughly 80%
α-humulene, β-myrcene, and β-caryophyllene, which explains their
location in the positive scores of PC1. Furthermore, these samples had
intermediate terpenic characteristics of hops grouped within the red and
black ellipses in the PCA, which justified placing these samples between
both groups. These samples showed both bittering and aromatic hop
characteristics. Some samples within this group presented a high
α-humulene content and high α-acid content, which illustrates their
dual-purpose features (samples 11, 13, and 18). Leonardi et al. (2013)
have already assigned Northern Brewer (sample 11) as a dual purpose
hop due to its α-acids and terpenic profile, which contribute to both
bitter and aroma characteristics in beer. In this sense, Magnum (sample
13) and Herkules (sample 18) varieties could also be classified as dualpurpose
hops, since their terpenic profile is very similar to Northern
Brewer variety, with a high content of α-humulene, and they also presented
high content of α-acids.
4. Conclusion
A simple and straightforward extraction method by an ultrasoundassisted
approach employing HEX as solvent provided excellent results
to assess the terpenic fraction of hops, which was well correlated to the
conventional hydrodistillation procedure showing no significant
6

L.M. Duarte et al.
Food Research International 138 (2020) 109768
Fig. 3. Profile of major terpenes in samples grouped in Principal Component Analysis (A – red ellipse; B – black ellipse; C – green ellipse; D – orange ellipse), also
showing the labeled α-acid content (below the number which identifies each hop sample) and α-humulene/β-myrcene and α-humulene/β-caryophyllene ratios. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
difference when the nonparametric Wilcoxon rank-sum test was performed
(p-value = 0.122). Terpenes extraction in the 25 samples
analyzed in this study occurred simultaneously in just 1 h by applying
this batch extraction method. Also, terpenes screening was performed by
GC–MS, and the major compounds identified in all varieties were
α-humulene, β-myrcene, and β-caryophyllene, with contents varying
from 20.20–67.64%, 2.22–45.30%, and 9.97–24.62%, respectively.
Finally, hop samples were studied in a multivariate way by PCA,
enabling to distinguish bittering, aromatic, and dual-purpose hops based
on their terpenic profile. Aromatic hops were high in α-humulene; bittering
hops presented a high β-myrcene content, and dual-purpose hops
were evidenced with a more complex and intermediate terpenic profile,
but could also be distinguished along PC1 and PC2. This methodology
employing a straightforward batch extraction protocol associated with
powerful instrumental and mathematical approaches, presents great
potential to be used for evaluating hops quality control, fraud investigation,
and traceability by the brewing industry and hop growers.
CRediT authorship contribution statement
Lucas Mattos Duarte: Conceptualization, Methodology, Software,
Formal analysis, Writing - original draft. Tatiane Lima Amorim:
Conceptualization, Methodology, Software, Formal analysis, Writing -
original draft. Richard Michael Grazul: Methodology, Resources,
Writing - review & editing. Marcone Augusto Leal de Oliveira:
Conceptualization, Validation, Resources, Writing - review & editing,
Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors acknowledge the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), INCTBio (FAPESP grant No.
2014/50867-3 and CNPq grant No. 465389/2014-7), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG); Rede Mineira
de Química (RQ-MG, CEX – RED-0010-14), MCT/FINEP/CT-INFRA 01/
2013-REF 0633/13 and Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES: PNPD 23038.007000-2011-70) for fellowships
and financial support.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
7

L.M. Duarte et al.
org/10.1016/j.foodres.2020.109768.
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