LWT - Food Science and Technology 141 (2021) 110905

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

A new perspective of a well-recognized raw material: Phenolic content,

antioxidant and antimicrobial activities and α- and β-acids profile of
Brazilian hop (Humulus lupulus L.) extracts
Tarsila Rodrigues Arruda a, Patrícia Fontes Pinheiro b, Pollyanna Ibrahim Silva a, Patrícia
Campos Bernardes a, *
a
Department of Food Engineering, Center of Agrarian Sciences and Engineering, Federal University of Espírito Santo, Alegre, Brazil
b
Department of Chemistry and Physics, Center of Exact, Natural and Health Sciences, Federal University of Espírito Santo, Alegre, Brazil

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

Keywords: Hop is a plant with several biological properties. Many factors affect both the presence and concentration of hop
Brazilian hop compounds, such as cultivar, location and growth conditions. Recently, the interest of hop cultivation in Brazil
Bioactive compound has grown despite the tropical weather and it has been possible to obtain hop completely cultivated in Brazilian
Hydroethanolic extract
soil. This study analyzed the chemical composition and verified the antioxidant and antimicrobial activities of
Antimicrobial potential
Brazilian hop hydroethanolic extracts in comparison to a commercially available hop cultivar. Brazilian hops
Soft resin
were a good source of flavonoids (5.72–12.82 mg CE/g dw), and presented a high content of phenolic com­
pounds, in particular the Canastra cultivar (35.1 mg GAE/g dw). The Brazilian cultivars showed interesting
antioxidant and antimicrobial proprieties. Gram-positive and Gram-negative bacteria were susceptible to hop
hydroethanolic extracts and Staphylococcus aureus was inhibited by the extracts of all cultivars. The extracts
presented antifungal activity against Byssochlamys nivea and significant growth inhibition at the higher con­
centration tested (20 μl/ml). The hydroethanolic extracts exhibited a high content of α- and β-acids, mainly the
Vitoria, Columbus and Canastra cultivars, reaching almost 60% (w/w). These characteristics evidenced the
potential application of Brazilian hops as raw materials in both pharmaceutical and food industries.

1. Introduction mainly of flavonoids, and some of them are exclusive to hop in­
florescences, such as xanthohumol (Sanz et al., 2019). The polyphenols
Hop (Humulus lupulus L., Cannabaceae) is a worldwide crop mainly exhibit a wide variety of physiological proprieties, with multiple ap­
used in beer production. Most substances related to plant biological plications, specially the use as natural preservatives (Hrncic et al.,
proprieties are extracted from hop cones (Zanoli & Zavatti, 2008). 2019). The biological activities of different hop cultivars have aroused
According to both presence and concentration of hop chemical interest in this crop, and some studies have investigated the benefits of
compounds, a series of biological proprieties can be verified in greater or hops in food production, such as in meat matrices (Kramer et al., 2015),
lesser intensity, such as the antioxidant, antimicrobial and anti- in cheeses (Larson et al., 1996) and bread (Nionelli, Pontonio, Gobbetti,
inflammatory potentials, and anti-mutagenic, anti-allergic, neuro­ & Rizzello, 2018) especially due to its antimicrobial and antioxidant
protection, and estrogenic proprieties (Hrncic, Spaninger, Kosir, Knez, & potentials.
Bren, 2019; Sanz, Torres, Vilariño, & Domínguez, 2019). The α-acids (humulones) and β-acids (lupulones) represent a great
The hop chemical composition varies considerably with several portion of the dry constituents of hop cones and they are associated with
factors, such as cultivar, planting location, maturation and weather flavor and bitterness of beer, besides playing an important role in plant
during its growth (Abram et al., 2015). Phenolics, essential oils and α- properties, including antimicrobial potential (Sanz et al., 2019). The
and β-acids are the hop compounds of major industrial interest. main α-acids are cohumulone, humulone and adhumulone, which are
The phenolic fraction of hops (4%–14% dry hop) is constituted isomerized to iso-α-acids under high temperature conditions. Iso-α-acids

* Corresponding author. Department of Food Engineering, Federal University of Espírito Santo, Campus Alegre, Alto Universitário, s/n, P.O Box 16, 29500-000,
Alegre, Espírito Santo, Brazil.
E-mail addresses: [email protected], [email protected] (P.C. Bernardes).

https://doi.org/10.1016/j.lwt.2021.110905
Received 23 July 2020; Received in revised form 11 January 2021; Accepted 12 January 2021
Available online 21 January 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
T.R. Arruda et al. LWT 141 (2021) 110905

are considered the main agents of beer bitterness, also exhibiting anti­ Folin-Ciocalteu reagent according to Singleton and Rossi (1965) with
bacterial activity, mainly against Gram-positive bacteria (Kramer et al., modifications. An aliquot of 0.6 ml of each hydroethanolic extract pre­
2015; Steenackers, De Cooman, & De Vos, 2015). Furthermore, the main viously diluted with 96% ethanol (1:5, v/v) was added of 3.0 ml of
β-acids correspondents are colupulone, lupulone and adlupulone, Folin-Ciocalteu 1:10 (v/v) (Sigma-Aldrich, USA). After 3 min resting in
constituting approximately 10% of the hop dry weight and playing a the dark, 2.4 ml of Na2CO3 saturated solution (0.075 g/ml) were added.
potential role in plant characteristic flavor and antimicrobial activity The absorbance was determined after 1 h against the blank (96%
(Formato, Gallo, Ianniello, Montesano, & Naviglio, 2013; Larson et al., ethanol, v/v), at 760 nm in a spectrophotometer (Thermo Fisher Sci­
1996). entific, EVO300 PC, EUA). The total phenolic content was determined
Traditional methods and emergent technologies are being used to using a calibration curve of gallic acid (0–170 mg/l) (Sigma-Aldrich,
optimize the extraction of hop compounds. Extraction and purification USA), and a linear correlation (0.0093x – 0.0206; R2 = 0.9927) was
techniques interfere in the chemical composition of the extracts. The use obtained. The results were expressed as equivalents of gallic acid in
of organic solvents, such as ethanol, is a method commonly used in the milligrams of gallic acid equivalents per gram of dry sample (mg GAE/g
industry (Hrncic et al., 2019). dw).
In Brazil, hops are majorly imported, which results in a high crop cost
and, consequently, higher cost of the final product. Despite attempts to 2.4. Determination of total flavonoids
adapt the species in Brazil, specially aiming largescale production,
studies on the chemical and biological proprieties of Brazilian hops are Total flavonoid content was determined by spectrophotometry ac­
scarce. Due to the absence of information about Brazilian hops, this cording to (Kim, Jeong, & Lee, 2003), with modifications. From each
work established the chemical composition and investigate the antiox­ hydroethanolic extract, previously dilluted in 96% ethanol (1:5 v/v), a
idant, antibacterial and antifungal activities of hop extracts. 0.5 ml aliquot was collected and then added 2 ml destilled water and
For this purpose, hydroethanolic extracts were obtained from cones 0.15 ml 5% NaNO2 solution. After 5 min, 0.15 ml 10% (v/v) AlCl3 was
of nine Brazilian hop cultivars. These extracts were analyzed in terms of added and, 1 min later, 1 ml 1 mol/l NaOH was added to the mixture.
the total phenolic and flavonoid contents, and the antioxidant and Then, 1.2 ml of distilled water was added and the mixture was agitated
antimicrobial activities. Moreover, uPLC analysis identified the main α- by 30 s. The absorbance was measured at 510 nm. The content of total
and β-acids. flavonoids was determined using a standard catechin curve (0–140
mg/l) (Sigma-Aldrich, USA), and a linear correlation (0.0021x – 0.0101;
2. Materials and methods R2 = 0.9952) was obtained. The blank was prepared as previously
described, without the addition of AlCl3. The results were expressed as
2.1. Plant materials equivalents of catechin in milligrams of catechin equivalents per gram of
dry sample (mg CE/g dw).
The plant material consisted in cones of nine Brazilian hop cultivars:
Spalt, Saaz, Vitória, Cascade, Mantiqueira, Columbus, Brasylijsk, Can­ 2.5. Determination of antioxidant activity
astra and Halertau Mittelfrüh. Cascade, Mantiqueira and Brasylijsk are
new cultivars, originated in Brazil. The other cultivars were imported Hop cone extracts antioxidant activity was determined in vitro using
seedlings cultivated in Brazilian soil. The cultivars refer to 2017/2018 the radicals ABTS (2,2-azino-di-(3-ethyl-benzothialozine-sulphonic
harvest (September/2017 to July/2018), from Bom Jardim (Nova Fri­ acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) as described previously
burgo, Rio de Janeiro, Brazil, 22◦ 9′ 36′′ S, 42◦ 26′ 12′′ W) at a 690 m by Re et al. (1999) and Brand-Williams, Cuvelier, and Berset (1995),
altitude. The soil conditions in this location, known as “red-yellow respectively. In these assays, high values of antioxidant activity indicate
argisol,” have an acid pH of 4.6, high levels of potassium and low levels that the sample is rich in compounds reactive to the specific radicals, by
of calcium, magnesium and phosphorus. Mantiqueira cultivar, on the reduction reactions through electron transfer (Chaves, Santiago, & Alías,
other hand, was obtained from São Bento do Sapucaí (Serra da Man­ 2020).
tiqueira, São Paulo, Brazil, 22◦ 62′ 53′′ S, 45◦ 58’ 00′′ W) at 1550 m Briefly, the cation ABTS + solution was prepared by the addition of 7
altitude. This cultivar was harvested during the same period previously mmol/l ABTS (Sigma-Aldrich, United States) to 2.45 mmol/l K2S2O8, at
mentioned and the soil conditions are characterized by high levels of a 1:1 (v/v) ratio. The mixture was mixed and allowed to stand in the
organic matter (lowland soil). The climate of the locations where the dark, at room temperature, for 16 h. The solution containing the ABTS
hop cones were obtained, high-altitude subtropical, is characterized by radical cation used in the experiment was diluted with ethanol to
low temperatures during winter (5 ◦ C, or even lower) and averages of absorbance 0.700 (±0.02), at 734 nm. For the analysis, 3.5 ml of the
28 ◦ C during summer days. radical ABTS + solution was added to 0.5 ml of the hydroethanolic hop
The hop cones were previously air dried at 45 ◦ C for 5 h, vacuum cone extracts diluted 1:50 in 96% ethanol. After reacting for 6 min, the
packed and stored at − 12 ◦ C until further analysis (approximately 7 absorbance was measured in a spectrophotometer at 734 nm.
days). A commercial cultivar (Premiant, Czech Republic), in pellets, was To the DPPH (Sigma-Aldrich, USA) assay, a 1 mmol/l radical solu­
also analyzed. tion was prepared, and the absorbance was adjusted to 0.700 (±0.02) at
515 nm. A 0.5 ml aliquot of each hop cone extract diluted in 96%
2.2. Production of hop extracts ethanol (1:5 v/v) was added of 3.5 ml DPPH radical solution. After 30
min in the dark, the absorbance was measured at 515 nm.
Hop cones were macerated with a mortar and pestle, and 500 mg Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
from macerated samples were homogenized with 25 ml 96% (v/v) (Sigma-Aldrich, USA) was used as a standard antioxidant. Trolox cali­
ethanol (Analítica, Brazil). The extractions were conducted at 60 ◦ C for bration curves were established (0–110 μmol/l) and the recorded ab­
24 h in a water bath (Tecnal, Brazil). The extracts were cooled to room sorbances of hop cones hydroethanolic extracts from each assay were
temperature and then centrifuged at 2470 g for 10 min (Excelsa II, inserted in their respective linear equations: Abs = − 0.005x + 0.5512
FANEM, Brazil). The supernatants obtained were stored at − 12 ◦ C until (R2 = 0.9991) for ABTS and Abs = − 0.0036x + 0.5038 (R2 = 0.9925) for
analysis (Abram et al., 2015). DPPH.
Where Abs is the absorbance recorded and x is the concentration (in
2.3. Determination of total phenolics μmol/l of Trolox equivalents) of diluted hydroethanolic hop cone ex­
tracts. The antioxidant activity of each hop cone extract was calculated
Total phenolics were determined by spectrophotometry using the considering the dilution factors (1:50 for ABTS and 1:5 for DPPH) and

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T.R. Arruda et al. LWT 141 (2021) 110905

sample weights. Results for ABTS and DPPH analysis were expressed in 2.7. Determination of α- and β-acids
μmol of Trolox equivalent per gram of dry weight (μmol Trolox/g dw).
Determination of α- and β-acids in the hops hydroethanolic extracts
2.6. Antimicrobial activity were made through chromatographic analysis using uPLC system
equipped with a quaternary pump system and DAD detector (Waters
Antimicrobial activity was tested against five foodborne pathogenic ACQUITY H-Class PLUS, Waters Corporation, EUA). The extracts were
bacteria, divided in Gram-positive, Staphylococcus aureus (ATCC 6538) previously filtered in 0.22 μm membrane. A reverse phase column C18
and Listeria monocytogenes (ATCC 7644); and Gram-negative, Escherichia (50 mm × 2.1 mm, 1.7 μm) (Waters Corporation, Ireland) was used. The
coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 15442), and Salmo­ separation was run at 30 ◦ C with a 0.2 ml/min flow rate. The gradient
nella Typhimurium (ATCC 14028). The antifungal activity was tested elution consisted of mobile phase A, composed of acetic acid (Dinâmica,
against Byssochlamys nivea (isolated from fruit juice). Brazil) in water (25:975, v/v), and mobile phase B (acetonitrile,
Cultures were stored at − 80 ◦ C. Bacteria were activated twice in BHI Dinâmica, Brazil) and the following gradients of 0–20 min, 100%–0% A
(Brain Heart Infusion) broth (Himedia, India) and cultivated in PCA in B, 20–27 min, 100% B (Abram et al., 2015). The injection volume was
(plate count agar) (Himedia, India) for 24 h at 35 ◦ C. B. nivea inoculum 3 μl, and the wave length used for detection was 320 nm. A mixture of
was obtained from a spore suspension, and a small sample was used to α-acids and β-acids (Versuchsstation Schweizerische Brauereien,
activate the cells in nutrient broth (Himedia, India) at 35 ◦ C for 24 h. Switzerland) of known composition, ICE-3 (International Calibration
Standard: 13,88% cohumulone, 30,76% N + adhumulone, 13,44%
2.6.1. Bacterial susceptibility test colupulone, 10,84% N + adlupulone) was used as an external standard
Bacterial susceptibility assay was performed using the agar diffusion to quantify compounds. The contents of α- and β-acids were calculated
method, as described by Deans & Ritchie (1987), with modifications. using standards calibration curves (0.075–9.6 mg/ml) and the results
Mueller-Hinton agar (Sigma-Aldrich, India) plates were inoculated with are given in dry weight: 3145396.77x – 456462 to cohumulone, with R2
a swab dipped into a bacterial solution with a turbidity equivalent to 0.5 = 0.9973; 6670991.14x – 234732 to humulone + adhumulone, with R2
McFarland standard (Probac, Brazil). A 6 mm diameter hole was made in = 0.9994; 2509653.55x – 45885 to colupulone, with R2 = 1; and
each inoculated plate center. A 5 μl aliquot of each extract was sepa­ 2350128.32x – 130179 to lupulone + adlupulone, with R2 = 0.9936.
rately added to the agar cavities. The antibiotics tetracycline (Inlab,
Brazil) and ampicillin (Inlab, Brazil) were used as positive controls, and 2.8. Experimental design and statistical analysis
96% (v/v) ethanol was used as a negative control. The plates were
incubated at 35 ◦ C for 24 h and the inhibition zones were measured in The experimental design was completely randomized and the ex­
millimeters. The diameter of the inhibition zones indicates the antimi­ periments were evaluated in triplicate and in three independent repli­
crobial activity of the extracts: higher inhibition zones diameter in­ cates, except for antimicrobial tests, in duplicate. The results were
dicates a higher antimicrobial activity of the hydroethanolic extract. submitted to analysis of variance (p < 0.05) and multiple comparative
tests when pertinent. On the antifungal activity, the analysis of variance
2.6.2. Minimum inhibitory concentration and minimum bactericidal was conducted and followed by linear regression (p < 0.05) (SigmaPlot
concentration 11.0, Systat®). The software Statistica version 12.0 (StatSoft®) was
Minimum inhibitory concentrations (MICs) of the extracts were employed in the statistical analysis.
determined using the broth microdilution test in 96-well microplates as
previously described by Bassanetti et al. (2017) with modifications. 3. Results and discussion
Aliquots of 100 μl BHI broth were added to a microplate well. Then, 100
μl of each extract were transferred to the wells of the first row and 3.1. Phenolic profile and antioxidant activity
serially diluted. For inoculum preparation, selected colonies of each
individual pathogenic bacteria were suspended in 0.85% (w/v) saline The incentive for promoting the cultivation of hops in Brazil has been
solution until obtaining solutions with turbidity equivalent to 0.5 increasing for the past five years, and studies on the chemical compo­
McFarland standard (108 CFU/ml). The suspensions were twice diluted, sition and biological activities of hops cultivated in tropical weather
resulting in a 106 CFU/ml inoculum. Aliquots of 100 μl of the obtained countries are scarce.
inoculum were added to the respective wells. The microplates were Among the hydroethanolic extracts investigated (Table 1), the one
incubated at 35 ◦ C for 24 h. MIC was defined as the lowest concentration originated by the Canastra cultivar showed the highest total phenolic
of hydroethanolic hop cone extracts (in mg/ml) that prevents visible content (35.10 mg GAE/g dw), while Spalt and Saaz cultivars exhibited
bacterial growth in microplate wells. For Minimal Bactericidal Con­ the lowest content (15.22 and 14.31 mg GAE/g dw, respectively). In
centration (MBC) determination, aliquots of 100 μl from each well contrast, the hydroethanolic extracts of Premiant (13.08 mg CE/g dw)
without visible growth (MIC), and from the well immediately adjacent and Columbus (12.82 mg CE/g dw) cultivars showed the highest con­
were inoculated onto BHI agar (Himedia, India) and incubated at 35 ◦ C, tents of total flavonoids (Table 1).
for 24 h. MBC was defined as the lowest concentration of hydroethanolic It is known that factors such as cultivar, hop growing location and
hop cone extracts (in mg/ml) required to kill a bacterium, evidenced by weather conditions affect the content of secondary metabolites in plants,
the absence of bacterial growth after inoculation (Bassanetti et al., influencing the accumulation of different compounds, such as hop
2017). phenolics, and among them flavonoids (Abram et al., 2015). In this
work, the cultivar seemed to be a significant factor in the variation of the
2.6.3. Antifungal activity concentration of phenolic compounds in hop cones since the samples
Antifungal assay against B. nivea was determined using the meth­ that exhibited the highest and lowest contents of phenolic compounds
odology described by Pires and Piccoli (2012), with modifications. Petri were harvested from the same area, and grown under the same
dishes were filled with 10 ml of PDA (potato dextrose agar) (Sigma-Al­ conditions.
drich, Spain). After solidifying, 10 ml of PDA homogenized with A study by Kowalczyk, Micha, Cichocka, and Gawlik-dziki (2013)
hydroethanolic hop cone extracts in different concentrations (0, 0.4, 1, evaluated, beyond cultivar, the extraction method. The hydroethanolic
5, 10, and 20 μl/ml) were poured onto the first layer of PDA. A platinum extracts (50% v/v ethanol) of hop cones from Magnum (Germany) and
wire loop was used to transfer a small sample of B. nivea inoculum to the Marynka (Poland) varieties showed higher contents of phenolic com­
center of the Petri dish. Plates were incubated at 25 ◦ C for seven days, pounds (44.22 and 54.78 mg GAE/g dw, respectively), when compared
and the mycelial diameter was measured in millimeters. to the Brazilian cultivars. However, the hop cultivars produced in Brazil

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Table 1 In addition, although the average temperature in hop planting lo­

Total phenolic content (TPC), total flavonoid content (TFV) and antioxidant cations is lower than the ones found in other regions of tropical countries
activity (AA) of hop extracts (mean ± SD, n = 3). during summer (in Brazil, between December and March), temperatures
Cultivar TPC (mg TFV (mg CE/ AA (μmol Trolox/g dw) at these growing areas are higher than the ones found in subtropical
GAE/g dw) g dw) countries, such as in North America and Europe. These higher temper­
ABTS DPPH atures might enhance plant stress in hops, which might be responsible
a for the increase secondary metabolites production in plants, such as
Spalt 15.22 ± 0.70 6.70 ± 0.72 58.21 ± 27.52 ±
f e 18.17 d 7.99 ab polyphenols. This is one of the hypotheses that might justify the high
Saaz a
14.31 ± 0.69 5.72 ± 0.49 56.91 ± 25.57 ± content of total phenolic compounds and, consequently, the high anti­
f e 21.96 d 4.19 b oxidant activity of Brazilian cultivars.
a
Vitória 29.34 ± 0.23 10.56 ± 132.45 ± 24.11 ± The new Brazilian cultivars, Mantiqueira, Cascade and Brasylijsk,
c 0.90 bc 15.19 abc 2.03 b
Cascadea 21.78 ± 0.39 7.52 ± 0.54 79.24 ± 29.24 ±
were not among the cultivars possessing the higher concentration of
e e 13.21 cd 5.80 ab total phenolics and, consequently, did not stand out for their antioxidant
Mantiqueiraa 20.18 ± 0.60 10.46 ± 78.53 ± 26.86 ± potential compared to other hop varieties grown in Brazil. Nevertheless,
e 0.09 bc 10.89 cd 2.80 b the antioxidant activity of the extracts obtained from these cultivars is
a
Columbus 31.09 ± 0.26 12.82 ± 139.84 ± 29.95 ±
similar to the one exhibited by the hydroethanolic extract from com­
bc 1.30 ab 25.44 ab 4.29 ab
Brasylijska 20.46 ± 0.38 7.98 ± 0.60 65.98 ± 25.75 ± mercial cultivar Premiant. Furthermore, the flavonoid content of the
e de 11.85 d 4.48 b extracts produced from Mantiqueira cultivar (grown at 1550 m) is
Canastraa 35.10 ± 0.19 10.13 ± 165.13 ± 35.15 ± higher than the one observed for Cascade and Brasylijsk cultivars
a 0.12 cd 2.97 a 4.87 ab (grown and 690 m). This result can be attributed, in addition to the
Halertau 31.38 ± 1.21 10.56 ± 90.16 ± 43.66 ±
Mittelfrüha b 0.66 bc 22.67 bcd 11.62 a
cultivar aspect, to plant location of Mantiqueira.
Premiantb 24.96 ± 0.69 13.08 ± 84.10 ± 25.48 ± It is relevant to mention that during the pelleting process hops are
d 1.45 a 34.60 bcd 1.17 b exposed to air and temperatures above 55 ◦ C. However, according to
a
Brazilian hop cultivar.
Krofta, Mikyška, and Hašková (2008), pelleting temperature did not
b
Cultivar from Czech Republic. Means with a different letter within columns have a significant effect on the phenolic content and on the antioxidant
differ significantly by Tukey’s test (p ≤ 0.05). action of hops. Also, the temperature used in the pellet production is
similar to the ones used in the extraction process performed in this
were a better source of flavonoids than the European cultivars. A wide study, which does not represent an important factor to elucidate the
range of chemical constituents represents the phenolic fraction of hops, differences exhibited by hops constituents in pellets and bloom.
including flavonoids. In addition to the cultivar factor, differences in the
chemical composition of hops from different countries are probably 3.2. In vitro antimicrobial tests
related to climatic conditions and soil composition.
Alternatively, the total phenolic content of hop methanolic (80% v/v 3.2.1. Bacterial susceptibility
methanol) extracts (23.1 mg GAE/g dw) obtained by Kähkönen et al. The bacterial susceptibility to hydroethanolic extract from hop cones
(1999) with ultrasound aid was smaller than the one obtained from was verified by the agar diffusion method using cavities containing the
some cultivars investigated in this work. This fact highlights the influ­ extract. The extracts exhibited distinct activities against the tested
ence of cultivar, planting location and employed extraction techniques bacteria and with S. aureus is the microorganism most sensitive to the
and demonstrates that Brazilian cultivars are a good source of phenolic hydroethanolic extracts (Table 2).
compounds. The hydroethanolic extract from the Columbus cultivar showed
The antioxidant assay using ABTS and DPPH radicals highlights the similar activity to the one presented by the control antibiotics against
capacity of an antioxidant to prevent the action of reactive free radical S. aureus, with a 35.5 mm inhibition zone, compared to tetracycline
species with the components of biological systems (Silva et al., 2018). (38.8 mm) and ampicillin (40.8 mm). Furthermore, comparing the re­
According to DPPH results, the antioxidant activity of the extracts in this sults between different cultivars, just the Brazilian Spalt exhibited ac­
study ranged between 24.11 and 43.66 μmol Trolox/g dw. The hydro­ tivity against all tested bacteria, with similar inhibition zones to all five
ethanolic extract obtained from Halertau Mittelfrüh cultivar imported microorganisms. In general perspective, E. coli, P. aeruginosa and
seedlings, but grown in Brazil at 690 m on the red-yellow argisol soil, L. monocytogenes seemed to be less susceptible to hop hydroethanolic
presented an antioxidant potential superior to the one found in the extracts, exhibiting inhibition zones against only four of the ten tested
extract produced from the commercial cultivar used as comparison cultivars.
parameter (Premiant). In the data obtained from ABTS assay, extracts Weber et al. (2019) analyzed the potential of supercritical
from Vitória (132.45 μmol Trolox/g dw), Columbus (139.84 μmol Tro­ hop–CO2–extracts (with 50% humulone and lupulone) incorporated into
lox/g dw), and Canastra (165.13 μmol Trolox/g dw) cultivars exhibited gels for topical use against Propionibacterium acnes and S. aureus and
the greatest antioxidant potentials, values even superior to the ones of verified inhibition zones of 5.5 mm and 3.0 mm, respectively. In a
the Premiant commercial variety extract. The different results obtained similar study, evaluating the isolated antibacterial activity of some hop
in both assays are related to the distinct affinity of radicals to molecules components, Bhattacharya, Virani, Zavro, and Haas (2003) observed
and due to the higher stability, and thus less reactiveness, of DPPH inhibition zones of 13 mm and 10 mm to β-acids and xanthohumol,
radical compared with ABTS (Mareček et al., 2017). respectively, when tested against Streptococcus mutans.
The phenolic compounds (flavonoids and phenolic acids) are some of The rich chemical composition of hop cones is responsible for its
the main agents responsible for the antioxidant activity of hops, being antimicrobial activity. Lupulones, humulones and xanthohumol are
the varieties with a higher content of phenolics associated with a greater identified as the main compounds associated with this activity (Alon­
antioxidant potential (Kowalczyk et al., 2013; Sanz et al., 2019). Des­ so-Esteban et al., 2019). Moreover, other substances such catechins and
methylxanthohumol and xanthohumol, prenylflavonoids exclusive to other hops phenolic compounds are described as potential antimicro­
hop, are also related to the plant antioxidant proprieties (Bocquet et al., bials (Gomes et al., 2018).
2019). Therefore, the differences in the concentration of these com­ The hydrophobic nature of hop antimicrobial agents facilitates its
pounds found in each hop cultivar results in singular antioxidant penetration in the bacterial cell wall and the interaction with the inner
potentials. membrane, resulting in damage to the cell structure (Rozalski et al.,
2013). In this context, the inhibition occurs in the active transport of

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T.R. Arruda et al. LWT 141 (2021) 110905

Table 2
Mean diameter (mm) of inhibition zones for the extracts obtained from different hop cultivars towards five pathogenic bacteria (mean ± SD, n = 3).
Cultivar Bacteria

S. aureus E. coli P. aeruginosa S. Typhimurium L. monocytogenes

Spalt 18.2 ± 2.9 eA 10.8 ± 5.0 bA 10.5 ± 2.8 bA 11.5 ± 1.4 bA 15.2 ± 3.2 abA
Saaz 20.8 ± 1.7 cdeA 11.3 ± 1.9 bA 14.2 ± 3.2 abA 0.0 ± 0.0 cB 0.0 ± 0.0 bB
Vitória 29.7 ± 5.5 cA 14.7 ± 5.3 bB 10.0 ± 9.2 bB 0.0 ± 0.0 cC 18.2 ± 2.5 abC
Cascade 20.2 ± 3.2 deA 0.0 ± 0.0 cB 0.0 ± 0.0 cB 0.0 ± 0.0 cB 12.0 ± 0.0 bA
Mantiqueira 23.7 ± 10.7 cdeA 0.0 ± 0.0 cC 0.0 ± 0.0 cC 0.0 ± 0.0 cC 10.0 ± 3.5 bB
Columbus 35.5 ± 17.3 abcA 8.7 ± 1.1 bcB 0.0 ± 0.0 cB 9.7 ± 1.1 bB 0.0 ± 0.0 cB
Brasylijsk 27.7 ± 5.3 bcdA 0.0 ± 0.0 cC 0.0 ± 0.0 cC 10.0 ± 2.1 bB 0.0 ± 0.0 cC
Canastra 26.5 ± 0.9 cdA 0.0 ± 0.0 cB 0.0 ± 0.0 cB 9.5 ± 0.9 bcB 0.0 ± 0.0 cB
Halertau Mittelfrüh 14.7 ± 1.9 eA 0.0 ± 0.0 cB 0.0 ± 0.0 cB 12.5 ± 2.1 bA 0.0 ± 0.0 cB
Premiant 19.8 ± 0.8 eA 0.0 ± 0.0 cC 14.0 ± 2.1 abA 12.0 ± 6.4 bB 0.0 ± 0.0 cC
Tetracycline 38.8 ± 1.7 aA 27.3 ± 4.5 aB 20.5 ± 0.5 aB 31.2 ± 7.8 aAB 23.2 ± 7.3 aB
Ampicillin 40.8 ± 0.8 aA 18.2 ± 0.8 abB 19.0 ± 9.9 abB 20.7 ± 0.3 bB 17.8 ± 6.0 abB

Means with a different letter (a, b, c, d, e) within columns differ significantly by Duncan’s test (p ≤ 0.05). Means with a different letter (A, B, C) within rows differ
significantly by Duncan’s test (p ≤ 0.05).

sugars and amino acids (Teuber & Schmalreck, 1973). However, lupulone, xanthohumol and iso-xanthohumol.
greatest antibacterial activity of hop cones is verified against
Gram-positive bacteria, such as S. aureus and L. monocytogenes (Kramer 3.2.2. Minimal inhibitory concentration and minimal bactericidal
et al., 2015). concentration
The cell surface of Gram-positive bacteria is more hydrophobic The hop cone hydroethanolic extracts showed inhibitory activity
compared to that of Gram-negative bacteria (Lim & Mustapha, 2007), against all five bacteria evaluated in the study (Table 3), with MIC
which might explain its greater susceptibility to the hops nonpolar means ranging between 0.093 mg/ml and 7.21 mg/ml, meanwhile the
molecules that exhibit antibacterial activity. In addition, the lipopoly­ MBCs varied from 0.12 mg/ml to 8.65 mg/ml.
saccharides (LPS) components of the outer membrane of Gram-negative The antimicrobial effect against S. aureus, among the other micro­
bacteria acts as a barrier to several hydrophobic molecules (Delcour, organisms evaluated, is highlighted, requiring the lowest concentrations
2010; Donnenberg, 2015). The fraction of LPS oligosaccharides can be of hop cone extract to inhibit bacterial growth. The mentioned Gram-
extended up to 30 Å over the outer membrane surface, inhibiting the positive bacteria did not present visual growth at the lowest concen­
diffusion of substances such α-acids, β-acids, and polyphenols (Fattouch trations tested for the hydroethanolic extracts of the Spalt (MIC < 0.097
et al., 2007; Teuber & Schmalreck, 1973; Wiener & Horanyi, 2011). mg/ml), Vitória (MIC < 0.13 mg/ml), Cascade (MIC < 0.096 mg/ml),
Likewise, some acidic components of hop cones, such as iso-α-acids can Columbus (MIC < 0.11 mg/ml), Brasilisjk (MIC < 0.11 mg/ml) and
dissipate the transmembrane proton gradient and reduce the internal pH Canastra (MIC < 0.093 mg/ml) cultivars, requiring concentrations
of the microbial cells due to their ionophore properties (Schurr, Hahne, slightly higher for the bactericidal effect (MBC). Alternatively, among
Kuster, Behr, & Vogel, 2015). the tested bacteria, the Gram-negative S. Typhimurium showed the
The same assay used in this work to verify the bacterial susceptibility highest MIC and MBC for the hop hydroethanolic extracts. Furthermore,
to hop cones extract was proposed by Dušek, Jandovská, Čermák, all three new Brazilian cultivars, along with those cultivars from im­
Mikyška, and Olšovská (2016) against S. aureus to identify the bioactive ported seedling but grown in Brazil, demonstrated a greater antimicro­
compounds of hop cones extract responsible for the formation of inhi­ bial potential than the commercial cultivar against the bacterial species
bition zones. A group of approximately 56 compounds was related to the evaluated in the study, with the lowest MIC evidenced for the Canastra
antimicrobial activity, including cohumulone, humulone, colupulone, cultivar.

Table 3
Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC), in mg/mla, of hop extracts against five foodborne pathogens.
Hop cultivar Bacteria

Staphylococcus aureus Escherichia coli Salmonella Typhimurium Pseudomonas aeruginosa Listeria monocytogenes

Spalt MIC <0.097 0.81 1.29 0.81 0.81

MBC 0.16 2.58 5.17 2.59 2.59
Saaz MIC 0.36 0.87 0.87 0.87 0.87
MBC 0.69 2.89 3.46 2.31 3.46
Vitória MIC <0.13 0,89 1.07 0.89 1.07
MBC 0.13 2.85 2.85 2.85 3.56
Cascade MIC <0.096 0.77 0.77 0.64 0.77
MBC 0.16 2.56 3.08 2.05 3.08
Mantiqueira MIC 0.33 1.12 1.86 1.19 1.19
MBC 1.30 3.35 7.46 4.47 4.65
Columbus MIC <0.11 0.91 1.21 0.91 0.91
MBC 0.15 3.02 3.63 3.02 3.63
Brasilijsk MIC <0.11 0.89 1.18 0.89 0.89
MBC 0.15 2.96 3.56 2.96 3.70
Canastra MIC <0.093 0.74 0.99 0.62 0.74
MBC 0.12 2.48 2.98 2.98 2.98
Halertau Mittelfrüh MIC 0.08 0.46 0.61 0.38 0.46
MBC 0.096 1.53 1.83 1.53 1.83
Premiant MIC 0.18 5.77 7.21 1.98 3.24
MBC 0.36 7.21 8.65 6.49 7.21
a
Concentration of hop extracts (mg of hop cones per ml of each extract).

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T.R. Arruda et al. LWT 141 (2021) 110905

We think that phenolic compounds, specially the prenylated chal­ Table 4

cones, togheter with acylphloroglucinol derivatives (α- and β-acids) Means of mycelial diameter (mm) of Byssochlamys nivea towards each
have a special role in the anti-staphylococcal activity (Bocquet et al., extract obtained from hop cultivars (mean ± SD, n = 3).
2019). Such results were reported in studies that mentioned the fact that Cultivar Mycelium diameter (mm)
species of Staphylococcus sp. are more susceptible to polyphenols (Silva Spalt 37.26 ± 19.22 abc
et al., 2018; Szewczyk, Zidorn, Biernasiuk, Komsta, & Granica, 2016; Saaz 38.76 ± 20.47 ab
Yam, Shah, & Hamilton-Miller, 2006). Vitória 37.04 ± 19.48 abc
In addition to the chemical composition of hops, the different pro­ Cascade 35.43 ± 20.37 bc
Mantiqueira 37.61 ± 19.82 abc
portions of the constituents and how they interact with other com­
Columbus 34.04 ± 20.46 c
pounds present in the plant material (i.e. proteins, carbohydrates) can Brasylijsk 41.31 ± 19.36 a
affect the extraction yield and, consequently, the extract properties, Canastra 35.14 ± 18.92 bc
such as antioxidant and antimicrobial activities. Aspects inherent to Halertau Mittelfrüh 38.74 ± 20.39 ab
cultivar, growing area and weather may be crucial to determining the Premiant 40.75 ± 22.52 a

extent of bioactive compounds in hops. Means with a different letter within columns differ significantly by
In this study, L. monocytogenes, a Gram-positive pathogen, showed Duncan’s test (p ≤ 0.05).
MIC values similar or superior to the ones exhibited by Gram-negative
bacteria. Shimwell (1937) mentioned in his work that there are differ­
ences in the susceptibility to hop components between species of
Gram-positive bacteria, which can justify the distinct extract activities
against L. monocytogenes and S. aureus.
L. monocytogenes and S. aureus were among the bacteria analyzed by
Kramer et al. (2015) to evaluate the antibacterial activity of the hop
components (α-acids, β-acids, and xanthohumol). The authors also
verified the highest activity of hop components against S. aureus. Some
differences can also be observed between bacterial cell surfaces that
could influence the higher staphylococcal susceptibility, for example,
the presence of a greater number of proteins attached to cell wall of
L. monocytogenes, making it more hydrophilic (Cabanes, Dehoux, Dus­
surget, Frangeul, & Cossart, 2002). Also, the peptidoglycan structure of
L. monocytogenes cell wall is similar to the one described for many
Gram-negative bacteria, such as E. coli (Bierne & Cossart, 2007).
Lupulones and humulones in hop cones are also associated with its
antimicrobial activity. Lupulones are known for a better bactericidal
activity, by being less soluble in water and having a greater hydropho­
bicity. The number and length of the side chains of their molecules
justifies this less solubility, which increases its lipophilic character and
Fig. 1. Effect of the concentration of the hop extracts on Byssochlamys nivea
assists the contact with the bacterial cell membrane (Bocquet et al.,
growth; y = − 14.8841 + 70.5833e − 0.0629x, R2 = 0.9782; y: mycelial diameter
2018; Larson et al., 1996; Teuber & Schmalreck, 1973).
(mm); x: Extracts concentration (μl/ml); R2: determination coefficient.
Xanthohumol is another compound of great importance to the anti­
microbial potential of hops. Acting as an inhibitor of diacylglycerol
extract at a concentration of 20 μl/ml (2% v/v) could considerably
acyltransferase, being a potent obstacle to lipid metabolism. This ac­
tivity could affect significantly the composition and stability of the cell reduce fungus growth, inhibiting almost completely B. nivea. Engelson,
Solberg, and Karmas (1980) verified that concentrations between 1.5%
wall and the membrane of microorganisms (Rozalski et al., 2013; Sakai
et al., 2012). To clarify the antimicrobial results, further work evalu­ and 2.5% (v/v) can inhibit the growth of vegetative forms of fungus,
ating xanthohumol content of Brazilian hops must be performed. such as Aspergillus niger and Aspergillus glaucus.
As mentioned, the disruption of the cellular membrane is the first The antifungal effect of hop cone extracts on some fungal species,
event associated with the antimicrobial activity of different hop com­ such as Penicillium roqueforti, Aspergillus parasiticus and Aspergillus niger,
pounds. The events that follow involve depleting the proton motive was demonstrated by Nionelli et al. (2018). The study evidenced the
force, a drop of the intracellular pH, inhibition of the respiratory chain possible use to hop extracts as natural preservatives in bread making,
and the proteins, DNA and RNA synthesis (Behr & Vogel, 2009; Teuber promoting extended shelf life.
& Schmalreck, 1973). This information was used to investigate the Bocquet et al. (2018) identified the hop compounds responsible for
resistance of some microorganisms, especially the ones involved in beer its antifungal activity. The study revealed that just some chalcones and
spoilage (Behr, Gänzle, & Vogel, 2006; Sakamoto & Konings, 2003). some acylphloroglucinols derivatives showed antifungal activity, being
desmethylxanthohumol and cohumulone the main bioactive com­
3.2.3. Antifungal activity against B. nivea pounds. Most reported studies on antifungal activity, unlike for the
An antifungal assay was realized with the fungus B. nivea, evaluating antibacterial activity, humulone derivatives are more active than the
the effect of five concentrations of hop cone hydroethanolic extracts. lupulone derivates (Bocquet et al., 2018; Taylor, Mizobuchi, & Sato,
There was no significant interaction (p > 0.05) between the hop cultivar 1985).
and the concentrations of hop cone extracts in analysis. The antifungal mechanisms of the mentioned compounds are still
The lowest mycelial diameter of the fungus is associated with a unknown. However, it is supposed that they act in different ways due to
greatest antifungal activity of the extracts. This effect was observed for their distinct structures. The damage in the cell respiratory chain is
associated with cohumulones effect (Bocquet et al., 2018). Hop cone
the extracts of the Cascade, Canastra and Columbus cultivars (Table 4),
which presented an antifungal effect significantly higher to the one components (e. g., α- and β-acids) could also exhibit activity toward the
fungal cell walls (which have a hydrophobic surface) after interactions
exhibited by the commercial cultivar Premiant (p ≤ 0.05).
In parallel, the decay of the mycelial diameter of B. nivea was related with hydrophobins (Bocquet et al., 2018; Gow et al., 2017). Hydro­
phobins are small (ca. 100 amino acids) and moderately hydrophobic
to the increase in the concentration of the hop cone extracts (Fig. 1). The

6
T.R. Arruda et al. LWT 141 (2021) 110905

proteins produced by fungi used to promote the growth of cells on hy­ characterized by a higher content of β-acids.
drophobic surfaces (Rillig, 2004). Among the ten hop cultivars evaluated, Columbus hydroethanolic
extract presented the highest humulones content, 33.47% (w/w). This
fact can be associated with its antifungal activity, whose extract was
3.3. Determination of α- and β-acids
responsible for one of the highest effects toward the mycelial growth of
B. nivea. In contrast, the extract obtained from Canastra cultivar pre­
The chromatographic analysis was conducted with hop cone
sented the highest content of lupulones, representing 30.37% (w/w) of
hydroethanolic extracts evaluated in this study. This work is one of the
the hop cones dry mass. A high content of lupulones might justify the
pioneers in the quantification of α- and β-acids, also known as soft resins,
smallest MIC identified in this study against Gram-positive bacteria
in hop cones hydroethanolic extracts by uPLC. This technique provides
S. aureus.
several economic benefits (reduction in time, energy and quantity of
In a study by De Keukeleire et al. (2003), using the female in­
organic solvents used in the analysis), besides being the more sensitive
florescences of different hop cultivars, the Admiral cultivar (Belgium)
and precise technique compared with traditional HPLC (Jurková, Čejka,
showed about 17.95% (w/w) of humulones (cohumulone and humu­
& Olsovská, 2012).
lone + adhumulone), while 6.77% (w/w) of the Wye Target cultivar
Chromatograms can be observed in Figures S1-S10 of Supplementary
(Belgium) consisted at lupulones (copululone and lupulone +
Material (peaks identified as A, B, C, and D, such as the elution order).
adlupulone).
The separation of the compounds and its quantification were obtained
Since the Vitória, Columbus, and Canastra cultivars were obtained
from a single run. The compounds of interest were identified and eluted
from the same harvest and growing area, these factors must not be used
in sequence: cohumulone (16.8 min), humulone + adhumulone (17.5
to justify the differences in the content of soft resins on their hydro­
min), colupulone (19.4 min), lupulone + adlupulone (20.0 min). The ad-
ethanolic extracts. Again, cultivar played an important role in the
compounds are among the minority constituents of soft resins (Hrncic
chemical composition of hops, influencing the formation and accumu­
et al., 2019), being unrequired for the proposal of this work in the
lation of α- and β-acids. Hop cultivar is related to their genetic potential
isolation and quantification of humulone/adhumulone and
to synthesize certain substances, including soft resins. Soil composition
lupulone/adlupulone.
and climate conditions can also be mentioned as significative dictators
This work confirmed the influence of hop cultivar in the content of
of the composition and properties of the hop varieties. The results
humulones and lupulones (Table 5). Seven out of ten hydroethanolic
indicate that some Brazilian microregions, such as Nova Friburgo (Rio
extracts analyzed exhibited a total content of soft resins (total α-acids +
de Janeiro) and Serra da Mantiqueira (São Paulo), are suitable for
β-acids) in agreement with the literature, that describes values in the
planting hops with greater industrial potential.
range from 6% to 38% (w/w) (Sanz et al., 2019).
Humulones are indispensable compounds to the quality of hops used
The exceptions that exceed this interval were the Vitória, Columbus,
at brewing, providing the bitter taste and contributing to the foam sta­
and Canastra cultivars, with hydroethanolic extracts showing contents
bility. These characteristics are related to its isomerization to iso-α-acids
exceptionally higher than the ones related, accumulating a total content
under high temperature (100–130 ◦ C) and pH (8–10) (Hrncic et al.,
of α- and β-acids of 45.62% (w/w), 54.29% (w/w) and 60.18% (w/w),
2019; Zanoli & Zavatti, 2008). Alternatively, lupulones are predomi­
respectively. High contents of soft resins at hop hydroethanolic com­
nantly associated with the antimicrobial effect discussed in this study,
pounds were not yet reported. As observed at Table 5, all of the α- and
but also to beer flavor (Sanz et al., 2019).
β-acids analyzed in present work exhibited high contents in the three
In addition to the role played in beer production, hops soft resins can
mentioned cultivars.
be used in the production of other food matrices and medicines due to its
The hydroethanolic extracts of hop cones presented distinct chro­
biological proprieties. The antioxidant and antimicrobial potentials of α-
matographic profiles, evidencing differences at the chemical composi­
and β-acids are of great value in assisting food conservation (Kramer
tion depending on the cultivar (Figs. 1–10, Supplementary Material).
et al., 2015; Larson et al., 1996; Nionelli et al., 2018).
The peaks related to humulones and lupulones of Vitória, Columbus, and
Canastra extracts exhibited larger areas than those found for the other
4. Conclusions
cultivar extracts evaluated in this study. These hop cones hydroethanolic
extracts of Brazilian cultivars are an exceptional source of soft resins.
The observed characteristics in the hydroethanolic extracts of
Other chromatographic analysis must be conducted to verify the whole
different hop cultivars from Brazil, in special the high content of α- and
chemical composition of the hop cultivars.
β-acids indicate a promising application as a raw material for both food
In a general perspective, most extracts evaluated presented similar
and pharmaceutical industries. The extracts obtained from the Vitória,
proportions of α- and β-acids. Only the ones from Columbus and Bra­
Columbus, and Canastra cultivars exhibited a high content of soft resins.
sylijsk cultivars exhibited substantial differences for both groups of
The cultivars evaluated in this study showed a promising role in food
substances. The hydroethanolic extract from Columbus cultivar showed
preservation due to its antioxidant and antimicrobial activities. Also, the
a highest content of α-acids, meanwhile, the Brasylijsk cultivar was

Table 5
Content of α-acids and β-acids in hop hydroethanolic extracts (mean ± SD, n = 3).
Cultivar α-acids (% w/w) β-acids (% w/w)

Cohumulone Humulone + Adhumulone Total α-acids Colupulone Lupulone + Adlupulone Total β-acids

Spalt 2.66 ± 0.41 e 2.46 ± 0.78 g 5.12 2.19 ± 0.51 f 3.67 ± 0.38 g 5.86
Saaz 4.54 ± 0.28 d 5.23 ± 0.34 e 9.77 4.15 ± 0.24 e 6.03 ± 0.12 e 10.18
Vitória 12.35 ± 0.24 b 9.71 ± 0.19 c 22.06 12.08 ± 0.25 b 11.48 ± 0.27 b 23.56
Cascade 3.86 ± 0.08 d 3.11 ± 0.04 fg 6.97 3.67 ± 0.05 e 4.15 ± 0.08 g 7.82
Mantiqueira 2.46 ± 0.03 e 2.48 ± 0.04 g 4.94 1.82 ± 0.02 f 2.24 ± 0.02 h 4.06
Columbus 15.48 ± 0.38 a 17.99 ± 0.47 a 33.47 10.79 ± 0.20 c 10.03 ± 0.18 c 20.82
Brasylijsk 2.50 ± 0.08 e 3.42 ± 0.14 fg 5.92 4.99 ± 0.22 d 10.19 ± 0.14 c 15.18
Canastra 15.69 ± 0.33 a 14.12 ± 0.32 b 29.81 15.11 ± 0.10 a 15.26 ± 0.15 a 30.37
Halertau Mittelfrüh 7.21 ± 0.19 c 6.76 ± 0.18 d 13.97 4.89 ± 0.32 d 7.17 ± 0.30 d 12.06
Premiant 3.11 ± 0.18 e 4.01 ± 0.29 f 7.12 1.76 ± 0.35 f 4.94 ± 0.15 f 6.70

Means with a different letter within columns differ significantly by Tukeys’s test (p ≤ 0.05).

7
T.R. Arruda et al. LWT 141 (2021) 110905

identification of volatile compounds and other chemical constituents of De Keukeleire, J., Ooms, G., Heyerick, A., Roldan-Ruiz, I., Van Bockstaele, E., & De
Keukeleire, D. (2003). Formation and accumulation of α-acids, β-acids,
these cultivars, which are still poorly explored, would be of great in­
desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus
terest aiming its application as natural preservative and raw material to lupulus L.). Journal of Agricultural and Food Chemistry, 51(15), 4436–4441. https://
beer industry. doi.org/10.1021/jf034263z
Deans, S. G., & Ritchie, G. (1987). Antibacterial properties of plant essential oils.
International Journal of Food Microbiology, 5(2), 165–180. https://doi.org/10.1016/
CRediT authorship contribution statement 0168-1605(87)90034-1
Delcour, A. H. (2010). Outer membrane permeability and antibiotic resistance.
Biochimica et Biophysica Acta, 1794(5), 808–816. https://doi.org/10.1016/j.
Tarsila Rodrigues Arruda: Investigation, Writing - original draft.
bbapap.2008.11.005.Outer
Patrícia Fontes Pinheiro: Conceptualization, Resources, Writing - re­ Donnenberg, M. S. (2015). Enterobacteriaceae. In Mandell, douglas, and bennett’s
view & editing, Supervision. Pollyanna Ibrahim Silva: Resources, principles and practice of infectious diseases (Vol. 2, pp. 2503–2517). https://doi.org/
10.1016/B978-1-4557-4801-3.00220-4
Writing - review & editing, Supervision. Patrícia Campos Bernardes:
Dušek, M., Jandovská, V., Čermák, P., Mikyška, A., & Olšovská, J. (2016). A novel
Conceptualization, Resources, Writing - review & editing, Supervision, approach for identification of biologically active phenolic compounds in complex
Project administration, Funding acquisition. matrices using hybrid quadrupole-orbitrap mass spectrometer: A promising tool for
testing antimicrobial activity of hops. Talanta, 156–157, 209–217. https://doi.org/
10.1016/j.talanta.2016.05.018
Declaration of competing interest Engelson, M., Solberg, M., & Karmas, E. (1980). Antimycotic properties of hop extract in
reduced water activity media. Hournal of Food Science, 45, 1175–1178. https://doi.
org/10.1111/j.1365-2621.1980.tb06514.x
The authors declare no conflicts of interest. Fattouch, S., Caboni, P., Coroneo, V., Tuberoso, C. I. G., Angioni, A., Dessi, S., et al.
(2007). Antimicrobial activity of tunisian quince (Cydonia oblonga Miller) pulp and
peel polyphenols extracts. Journal of Agricultural and Food Chemistry, 55(3), 963–969.
Acknowledgments https://doi.org/10.1021/jf062614e
Formato, A., Gallo, M., Ianniello, D., Montesano, D., & Naviglio, D. (2013). Supercritical
fluid extraction of alfa and beta-acids from hops compared to cyclically pressurized
The authors thank the Brazilian Federal Agency for the Support and
solid – liquid extraction. The Journal of Supercritical Fluids, 84, 113–120. https://doi.
Evaluation of Postgraduate Education (CAPES) for granting scholar­ org/10.1016/j.supflu.2013.09.021
ships. Paulo Cordeiro is also gratefully acknowledged for donating the Gomes, F. M. S., da Cunha Xavier, J., dos Santos, J. F. S., de Matos, Y. M. L. S.,
samples of hop cultivars indispensable to the execution of this work (in Tintino, S. R., de Freitas, T. S., et al. (2018). Evaluation of antibacterial and
modifying action of catechin antibiotics in resistant strains. Microbial Pathogenesis,
memorian). 115, 175–178. https://doi.org/10.1016/j.micpath.2017.12.058, 2018.
Gow, N. A. R., Latge, J., Munro, C. A., De Groot, P. W. J., Hellingwerf, K. J., Klis, F. M.,
et al. (2017). The fungal cell wall: Structure, biosynthesis, and function. Microbiology
Appendix A. Supplementary data
Spectrum, 5(3), 3341–3354. https://doi.org/10.1128/microbiolspec.FUNK-0035-
2016.Correspondence
Supplementary data to this article can be found online at https://doi. Hrncic, M. K., Spaninger, E., Kosir, I. J., Knez, Z., & Bren, U. (2019). Hop compounds:
Extraction techniques, chemical analyses, antioxidative, antimicrobial, and
org/10.1016/j.lwt.2021.110905.
anticarcinogenic effects. Nutrients, 11(257), 1–37. https://doi.org/10.3390/
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