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187 November / December 2017 (Vol. 70)
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
https://doi.org/10.23763/BrSc17-18oladokun
Authors
O. Oladokun, S. James, T. Cowley, K. Smart, J. Hort and D. Cook
Dry-Hopping: the Effects of Temperature
and Hop Variety on the Bittering Profiles
and Properties of Resultant Beers
This paper reports the effects of dry-hopping at 4 and 19 °C for a low alpha versus high alpha hop variety on
the resulting profiles of non-volatile hop acids (humulinones, iso-α-acids, α-acids). In a dry-hopping
study conducted over 2 weeks, we found a significant increase in humulinone concentration driven principally
by hop alpha acid content and the duration of dry-hopping. Conclusive evidence of iso-α-acid losses during
dry-hopping (by adsorption onto spent hops) is presented, in addition to a significant increase in α-acid
concentrations, which was observed only for beers dry-hopped at 19 °C with the high alpha hop variety.
Measured beer parameters (especially at 19 °C) revealed an increase in pH, ABV (%), and a decrease in beer
density during dry-hopping – from which we conclude that further attenuation of beer occurred during
dry-hopping. The polyphenol content of beers was found to increase substantially with dry-hopping time,
whilst both temperature and hop variety were found to be significant factors determining the amounts of
polyphenols extracted. Finally, analysis of the spent hop slurry (recovered after 14 days of dry-hopping)
confirmed that the residual content of hop acids (α-acids, their oxidised derivatives and polyphenol content),
makes these materials – currently treated as waste – of potential value for re-use in the brewing process.
Descriptors: dry-hopping, humulinones, ‘hop-creep’, bitterness quality, spent hops, hop polyphenols
1 Introduction
There is no doubting the significant contribution of dry-hopping to
the renaissance of craft beer and the subsequent boom in sales
of craft beers around the world. On the face of it, dry-hopping
represents a relatively simple means of improving the flavour of
beer; for this, brewers add between 2–12 g/L of hops in the form
of cones or pellets into beer during fermentation or conditioning
for periods ranging from several days to weeks [23]. The added
hops can be left in the beer with no agitation (static dry-hopping)
or with agitation, using a pump or CO2 for example (dynamic dry-
hopping). Brewers can also add hop oil essences to beer to create
specific flavour characters that mimic dry-hopped flavours in their
product [6]. Although the basics of dry-hopping as described above
are agreed, there is no common approach to dry-hopping within
the brewing industry; with most breweries adopting dry-hopping
Olayide Oladokun, Joanne Hort, David Cook, International Centre for
Brewing Science, Bioenergy and Brewing Science Building, University
of Nottingham, School of Biosciences, Division of Food Science, Sutton
Bonington Campus, Loughborough, UK; Sue James, Trevor Cowley, Ka-
therine Smart, Anheuser-Busch InBev, Woking, UK; corresponding author:
david.cook@nottingham.ac.uk
practices based on product line, brewhouse volume and proces-
sing capabilities. The addition of hops to beer in the cold stages
of processing results in a cascade of changes to beer quality – the
implications of which most brewers and researchers-alike are still
attempting to decipher. Brewers often have to consider several
factors in relation to dry-hopping in order to produce beers with
consistent hoppy flavour. These include hop variety selection and
harvest date, rate of addition, oil content of selected hop, dry-
hopping temperature, and whether to adopt a dynamic or static
dry-hopping process [4, 11, 23]. Even the alcohol content of the
beer to be dry-hopped must be considered, in order to prevent the
extraction of unwanted hop vegetative materials into the finished
product due to the solvating power of ethanol [20]. The duration of
dry-hopping (contact time) is another crucial factor to control the
balance of compounds extracted from hops into beer. Due to the
hydrophobicity of some hop aroma compounds, dry-hopping for
prolonged periods can cause their partitioning out of beer back onto
spent hops. The losses or reduction in concentration of important
marker aroma compounds such as linalool has been reported
after prolonged dry-hopping [23]. Both high alcohol content and
prolonged contact time can increase the extraction of unwanted
vegetative materials, which often lead to the generation of ‘grassy’
flavours in dry-hopped beers.
The increase in aroma perceived in dry-hopped beers versus
conventionally hopped beers is thought to be due to elevated
levels of several volatile terpene compounds, hydrocarbons and
their derivatives e.g. linalool, myrcene, humulene, β-Citronellol
and geraniol [11, 22, 23]. The labile nature of these compounds
means that they are rarely present in beers that are not dry-hopped,
because they are easily lost to evaporation during wort boiling or
November / December 2017 (Vol. 70) 188
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
fermentation [6]. Furthermore, the presence of yeast during dry-
hopping adds an extra level of complexity to this process. Some
researchers have reported the biotransformation of certain volatile
hop compounds during and post fermentation (maturation), e.g., in
the conversion of geraniol into β-citronellol [21]. This means that
brewers must also decide whether to totally remove yeast from beer
before dry-hopping. The presence of yeast during dry-hopping may
offer other benefi ts – suspended yeast can metabolise dissolved
oxygen during dry-hopping, thereby protecting beer from oxidation
during the process [23].
Perhaps one of the unintended consequences of dry-hopping is
the effect this process has on perceived beer bitterness. Several
studies have shown an increase in both measured analytical (BU)
and perceived bitterness in dry-hopped beers [1, 12, 16, 23]. These
observations have been explained by the presence of oxidized
α-acids (humulinones), which are highly prevalent in dry-hopped
beers. Humulinones have been reported in leaf and pellet hops
[14]. They are readily soluble in beer and have a bitterness intensity
of 66 % relative to iso-α-acids [1]. The second explanation is that
volatile aroma compounds in hops can enhance the perception of
bitterness intensity, as well as modify bitterness quality, particularly
at low BU levels - as was demonstrated through the addition of hop
oils to beers of different analytical BU [18]. This observation is most
likely due to a multimodal interaction between the perception of taste
and aroma. The extraction of hop polyphenolic compounds into
beer during dry-hopping is another contributory factor to increased
bitterness perception in dry-hopped beers; these compounds are
widely accepted to contribute both bitterness and astringency to
beer depending on their molecular mass [3, 15, 19]. Dry-hopping
has also recently been reported to affect beer parameters, e.g. beer
pH, foam and International Bitterness Unit (IBU) readings deter-
mined by the spectrophotometric method [12–14]. To date, most
of the available studies on dry-hopping have focused on volatile
hop aroma compounds and the dynamics of transfer of these com-
pounds into beer during
dry-hopping. Conse-
quently, this study was
designed to investigate
the behaviour of non-
volatile compounds
which affect perceived
bitterness during dry-
hopping, as infl uenced
by temperature and
time for two hop va-
rieties. A time-course
experiment lasting 14
days was designed
to investigate the ef-
fects of dry-hopping
with a low alpha hop
(Hersbrucker, 3 % α)
and high alpha hop
variety (Zeus, 17 % α)
at both 4 & 19 °C, on
the composition of hop
acids (humulinones,
iso-α-acids, α-acids)
in dry-hopped beers, as well as in the spent hops left after dry-
hopping. We further investigated the impacts of dry-hopping on
the total polyphenol content of dry-hopped beers, and several
beer parameters, including pH, ABV (%) and beer density in the
resulting dry-hopped beers.
2 Materials and methods
2.1 Hops
The hops selected for this study (T90 pellets of the varieties
Hallertau Hersbrucker and Zeus) were purchased from Simply
Hops (Paddock Wood, Kent, UK). The hops were of 2015 crop
year and contained the following amount of oil and α-acids, re-
spectively: Hersbrucker (0.5–1 mL /100 g, 3 %) and Zeus (2.5–3.5
mL /100 g, 17 %). The precise α-acid content was determined in
a previous study [17].
2.2 Hop acid standards and chemicals
Iso-α-acid standard (ICE-3) containing trans-isocohumulone,
trans-isohumulone, trans-isoadhumulone (62.3 % w/w) was
purchased from Labor Veritas Co. (Switzerland). Humulinones
were synthesised in-house using the method previously detailed
[19]. Extraction chemicals (methanol, dichloromethane) and those
used in the polyphenol assays were purchased from VWR (UK).
2.3 Beer production
A 50 L all-malt brew was produced from lager malt in the pilot
brewery of the University of Nottingham. A single infusion mash
protocol at 68 °C was adopted. The wort was hopped with East
Kent Goldings hops (6.8 % α-acids) to achieve a target of 20
bitterness units (BU). Boil duration was 60 min and the post-boil
gravity was 1.040 (10 °P). The hopped wort was cooled to 15
°C and transferred into 4 separate 30 L capacity FastFerment™
conical fermenters, each containing 11 L of wort. The wort in each
fermentation vessel was fermented with Safl ager S-23 dried yeast
(11 g) from Fermentis, with fermentation conducted at 18 °C for 7
days. The resultant young beer was cooled to 3 °C for 3 days to
give base beers with a fi nal gravity of 1.008 (2 °P). The alcohol
content of the base beer was 4.32 % ABV and pH was 3.82. At the
end of maturation, the sedimented yeast collected in a collection
bulb at the bottom of each fermenter was discarded (Figure 1). All
of the fermenters were left at room temperature to equilibrate for
4 h before being set up for dry-hopping (Figure 1).
2.4 Dynamic dry-hopping set-up
T90 pellet hops (of either Hersbrucker or Zeus variety) were added
to the fermentation vessels at a rate of 4 g/L and dry-hopping took
place in temperature controlled rooms set at 4 and 19 °C. A dyna-
mic dry-hopping set-up was mimicked by inserting an overhead
stirrer fi tted with a paddle into the fermenters. The headspace of
each vessel was purged with N2 after hop addition (to displace
O2), before the commencement of dry-hopping. The stirrers in all
four fermenters were set to rotate at 200 RPM with the aid of a
tachometer (Figure 1).
Fig. 1 Dynamic dry-hopping apparatus
189 November / December 2017 (Vol. 70)
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
2.5 Sample collection time points
Samples were collected over a 14-day period. The first time point
samples were collected prior to the addition of hops and denoted
Day 0 samples. The base beer had a total bitterness concentration
18.3 mg/L of iso-α-acids by HPLC. Subsequent samples were coll-
ected a day later (Day 1), 3, 7, 10 and 14. Samples were collected
from the bottom of the fermenter (in the collection bulb) with the
aid of the isolation valve.
2.6 Determination of hop acids in fresh and spent
hops
A small quantity of each hop variety was first pulverised with
a blender. Then 1 g of each hop was transferred into a 50 mL
centrifuge tube. Methanol (10 mL) was added to extract hop acid
compounds. Samples were extracted on a roller bed for 1 h, and
upon completion centrifuged at 5000 RPM for 5 min to aid phase
separation. An aliquot of the supernatant was filtered through a
0.20 µM polytetrafluoroethylene (PTFE) membrane syringe filter
in readiness for hop acid analysis by RP-HPLC (as below).
For spent hops, a slurry of spent hops was recovered from each
fermentation vessel at the end of dry-hopping (Day 14) and filtered
to dryness through a crucible under vacuum. One gram of the dry
spent hop material was then subjected to extraction as described
above.
2.7 Determination of hop bitter acid compounds
The separation of hop acids (humulinones, iso-α-acids, α-acids,
β-acids) and their relative concentrations in hops and beer was
determined by RP-HPLC as previously described in Oladokun et
al. (2016). Hop acids were extracted from beer as follows: beer
sample (5 mL) was acidified with orthophosphoric acid (100 uL)
and extracted into isooctane (10 mL) on a roller bed for 30 min.
The isooctane extract was subsequently transferred into a glass
tube and evaporated to dryness under nitrogen. The residue
was reconstituted in acetonitrile (2 mL) and analysed by HPLC.
Samples were analysed in triplicate and hop acid concentrations
were acquired from calibration curves generated from external hop
acid standards prepared in the range of 1, 5, 10, 20, 40, 60 mg/L.
Humulinone standards were prepared in the concentration range
of 1, 10, 20, 40 and 80 mg/L.
2.8 Total polyphenol content
The total polyphenol content of dry-hopped beers was determined
according to the standard beer ASBC Beer-35 method. Beer sample
(10 mL) was mixed with a preparation of carboxymethylcellulose
(CMC, 1 %) and ethylenediamine tetra acetic acid (EDTA, 0.2 %)
(8 mL) in a 25 mL volumetric flask, then ferric ammonium citrate
(3.5 %, 0.5 mL) was added, followed by ammonium hydroxide so-
lution (33.3 %, 0.5 mL) with mixing after each addition. The solution
was made up to mark with Reverse Osmosis (RO) water and left
to stand at room temperature for 10 min. The absorbance of the
solution was taken at 600 nm and multiplied by 820 to give the
total polyphenol content in beer (mg/L). The assay was conducted
in triplicate for each sample.
2.9 Total polyphenol content of fresh and spent
hops by Folin-Ciocalteau method
Total polyphenol content of spent hops was determined using the
Folin-Ciocalteau colorimetric assay. Pulverised hops (1 g each) were
first extracted into dichloromethane (15 mL) on a roller bed for 30
min (separately twice), in order to remove hop acid compounds. The
dichloromethane extract was discarded and the hop residue was
subjected to further extraction in 70 % methanol in water (10 mL)
for 30 min on a roller bed. Extraction was repeated once more
and the combined extracts were used for total polyphenol content
determination in hops. Recovered spent hop slurry was dried as
described in Section 2.6 before extraction with dichloromethane and
70 % methanol in water as described above. For the colorimetric
assay, 20 µL of the methanolic extract was combined with water
(1.58 mL) and Folin-Ciocalteau reagent (100 µL) in a cuvette. The
mixture was mixed well and left for 5 min to react. Saturated sodium
carbonate (300 µL) was added and the whole mixture was mixed
and left at room temperature for 2 h. Absorbance readings were
taken for the samples at 765 nm. Total polyphenol concentration
was determined from a gallic acid calibration curve of 0, 100, 150,
250, 500 mg/L, and expressed as gallic acid equivalent per litre of
hop polyphenol extract (mg of GAE/L of extract).
2.10 Determination of beer density, % ABV and pH
The alcohol content, density and gravity of beer samples were
measured using an Anton Paar DMA 4500 coupled to an Alcolyzer
Plus instrument (Anton Paar, Austria). Beer pH was determined
with a pH meter (Metler Toledo). All measurements were made
in triplicate.
2.11 Statistical analysis
Statistical analysis was carried out with XLSTAT version 2017
(Addinsoft, Paris). A 3-way (hop variety, temperature and time)
Analysis of Variance (ANOVA) test was conducted to determine
significant impacts (P < 0.05) of these dry-hopping factors on each
of the measured analytical properties of the resulting beers.
3 Results and Discussion
3.1 Impacts of dry-hopping on the resulting profile
of hop acid compounds in beer.
3.1.1 Humulinones
The concentrations of humulinones measured in beers dry-hopped
with Zeus and Hersbrucker are presented in figures 2A and 2B
respectively. For the beers dry-hopped with Zeus, (Figure 2A), a
significant increase (p < 0.05) in the concentration of humulinones
was observed at both temperatures investigated over the course of
dry-hopping. The largest increase in humulinone concentration (58
mg/L) was observed after just 24 h of dry-hopping at 19 °C; and
although at 4 °C, the concentration of humulinones also increased
to 37 mg/L after 24 h, the temperature at which dry-hopping was
conducted was not a significant factor (p > 0.05) determining the
concentration of humulinones in dry-hopped beers. The maximum
November / December 2017 (Vol. 70) 190
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The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
concentration of humulinones measured in the beers dry-hopped
with Zeus did not change significantly (p > 0.05) after the third
day of dry-hopping.
In beers dry-hopped with the low α-acid hop variety (Hersbrucker
in Figure 2B), both temperature and duration of dry-hopping were
significant factors (p < 0.05) in the concentration of humulinones
found in the dry-hopped beers. The concentration of humulinones
increased significantly over 14 days, with higher concentrations
observed at lower dry-hopping temperatures. Maximum concen-
trations of 11 and 8 mg/L of humulinones were reached in the
beers after 14 days of dry-hopping at 4 and 19 °C, respectively.
The greater levels of humulinones found in the beers dry-hopped
with Zeus, relative to Hersbrucker indicates that the formation
of humulinones may be linked to levels of α-acids present in the
hop used for dry-hopping. Indeed, hop variety was found to be a
significant factor (p < 0.05) in the concentration of humulinones
observed in the dry-hopped beers, with greater concentrations of
humulinones attained in beers dry-hopped with Zeus hops. This
observation is in contrast to the similar levels of humulinones
measured in the hops themselves prior to dry-hopping (~ 0.29
%w/w) which further suggests that concentrations of humulinones
in dry-hopped beers are not solely related to their extraction from
hops. The dynamic dry-hopping process adopted may well have
contributed to greater humulinone formation (from α-acids) compa-
red to a static dry-hopping process. These results therefore suggest
that the formation of humulinones from α-acids during dry-hopping
is possible, depending on the adopted dry-hopping procedure,
temperature and α-acid content of hops. To understand the levels
of humulinones in commercial beers, a Popular IPA brewed in the
UK was purchased and analysed by HPLC. The selected beer is
marketed as a 40 IBU beer that is dry-hopped at 10 g/L, with six
different aroma hop varieties. HPLC analysis revealed that this
beer contained 35 mg/L of humulinones, 26 mg/L of iso-α-acids
and 3.21 mg/L of α-acids (Figure 3). Thus, the concentration of
humulinones in this particular beer was in fact higher than that
of iso-α-acids. Based on a bitterness contribution of 66 % from
humulinones, these compounds would contribute a calculated
bitterness of 23.1 mg/L to yield a total bitterness value of 49.1 mg/L
(humulinones (ppm) x 0.66 + iso-α-acids (ppm)). This represents a
47 % contribution from humulinones to the total bitterness of this
dry-hopped beer, and further reinforces the significant impact of
humulinones in offsetting the reduction in bitterness encountered
due to losses of iso-α-acids during dry-hopping.
3.1.2 Iso-α-acids
Iso-α-acids represent the major source of beer bitterness and are
derived from the isomerisation of α-acids. This thermally driven
process occurs traditionally on the hot side of the brewing process
(in the kettle) as opposed to during dry-hop-
ping. Losses of iso-α-acids in dry-hopped
beers have been reported [8, 13], and were
confirmed in the present study (Figure 4),
with significant (p < 0.05) reductions in
iso-α-acid concentrations being observed
during dry-hopping with both hop varieties
(Zeus and Hersbrucker). Our results further
showed that the principal drop in iso-α-acid
concentrations occurred after the first day
of dry-hopping, especially at 19 °C in both
hop varieties studied (Figure 4A & B).
In the beers dry-hopped with Zeus at 19 °C,
there was no further significant (p > 0.05)
drop in the concentration of iso-α-acids over
the course of dry-hopping after 24 h; whilst
a gradual decrease in the concentration
of iso-α-acids was observed at 4 °C. For
both hop varieties, the concentration of
Fig. 2 Changes in the concentration of humulinones over the
course of dry-hopping at 4 and 19 °C with (A) high alpha
Zeus and, (B) low alpha Hersbrucker
Fig. 3 Hop acid profile of a commercial craft beer produced in the UK. Humulinones:
cohumulinone (13.32), humulinone (15.03), adhumulinone (15.72); iso-α-acids:
trans-isocohumulone (18.20), cis-isocohumulone (18.85), trans-isocohumulone
(20.73), cis-isocohumulone & trans-isoadhumulone (21.41), cis-isoadhumulone
(22.27; humulones: cohumulone (23.40), humulone (27.04) and adhumulone (28.03)
191 November / December 2017 (Vol. 70)
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of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
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iso-α-acids at the end of the 14-day dry-hopping period was not
dependent on the dry-hopping temperature. Overall, the significant
loss (p < 0.05) of iso-α-acids observed was proportionately greater
with Zeus as compared to Hersbrucker (there was a 50 % drop in
iso-α-acid concentration in beers dry-hopped with Zeus compared
to 28 % in Hersbrucker).
3.1.3 α-acids
Although some studies have reported an increase in the α-acid
concentration of dry-hopped beers [12], the overall levels of α-acids
found in beer is also dependent upon upstream processing, e.g.
vigour and duration of boil in the kettle, as well as the type of hop
products utilised for bittering. For example, beers bittered with
pre-isomerised hop products do not contain residual α-acids,
whilst beers that have been conventionally bittered in the kettle
with hop cones or pellets [19] retain residual concentrations of
non-isomerised α-acids. Post-dry-hopping concentrations of
α-acids are presented in figures 5A and B. A significant increase
(p < 0.05) in the α-acid content of the dry-hopped beers was only
observed in the beers dry-hopped with high alpha Zeus at 19 °C.
This increase was gradual over the course of dry-hopping, with
a smaller increase observed at the lower dry-hopping tempera-
ture of 4 °C (Figure 5A). For the low alpha variety (Hersbrucker)
shown in figure 5B, there was no significant (p > 0.05) increase
in the concentration of α-acids in the dry-hopped beers over the
14 days of dry-hopping. In contrast to Zeus, the temperature at
which dry-hopping was conducted did not make a significant (p
> 0.05) difference to the levels of α-acids found in the beers dry-
hopped with Hersbrucker.
These results therefore suggest that the content of α-acids in dry-
hopped beers may be associated with the α-acid content of the
hop variety used for dry-hopping, and therefore the use of high
alpha hops for dry-hopping at higher temperatures may yield higher
residual α-acids in the final product. Furthermore, hop variety was
found to be a significant factor (p < 0.05) in the concentration of
α-acids in dry-hopped beers; higher α-acid concentrations were
observed in Zeus compared to Hersbrucker. Although α-acids
at concentrations below 14 mg/L are thought not to contribute
substantially to beer bitterness [7], a considerable impact on per-
ceived bitterness of beer from the combination of humulinones,
iso-α-acids and elevated residual α-acids cannot be discounted.
The concentration of β-acids did not increase in the dry-hopped
beers presumably due to their documented poor solubility in beer.
3.2 Impact of dry-hopping on the total polyphenol
content of beer
3.2.1 Hersbrucker
The total polyphenol content (TPC) of the dry-hopped beers was
determined, and for the beers dry-hopped with Hersbrucker, the
result is presented in figure 6A. In the base beer, i.e. Day 0 sample,
the average TPC measured was 240 mg/L. Upon dry-hopping at
19 °C and after just 24 h we observed a significant increase in TPC
to 330 mg/L, representing a 38 % increase. The TPC increased
further, to 49 % above starting levels after 3 days of dry-hopping,
and then remained fairly constant for the rest of the dry-hopping
Fig. 4 Changes in iso-α-acid concentrations over the course of
dry-hopping at 4 and 19 °C with (A) high alpha Zeus and,
(B) low alpha Hersbrucker
Fig. 5 α-acid concentrations over the course of dry-hopping at
4 and 19 °C with (A) high alpha Zeus and, (B) low alpha
Hersbrucker
period. Thus, the highest increase in total polyphenol content of
the dry-hopped beers was observed after 3 days. The effect of
dry-hopping temperature on total polyphenol content of dry-hopped
beers is also evident in figure 6. Overall, there was proportionately
less of an increase in TPC of the beers when dry-hopped at 4 °C,
with a gradual increase in total polyphenol content over the dura-
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of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
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tion of dry-hopping observed at this temperature, and reaching a
peak at around day 10.
3.2.2 Zeus
For the beers dry-hopped with Zeus (Figure 6B), the biggest
percentage increase in TPC occurred during the first 24 h of dry
hopping at both temperatures investigated. However, the increase
in total polyphenol content of the dry-hopped beers for Zeus was
lower than that observed in Hersbrucker. Thus, the level of hop
polyphenols extracted into beer during dry-hopping was clearly
impacted by hop variety, but could also be controlled by dry-hopping
at lower temperatures. To emphasise the impact of hop variety on
the TPC of dry-hopped beers at 19 °C, our results show a maximum
percentage increase of 34 % in total polyphenols in Zeus after 14
days of dry-hopping, which was lower than the increase observed
in Hersbrucker after just 24 h of dry-hopping (38 %). A maximum
increase of 15 % in total polyphenol content was observed when
dry-hopping at 4 °C for the Zeus hop variety; this value was com-
parably higher at 30 % for the Hersbrucker hop variety.
3.3 Impact of dry-hopping on beer analytical
parameters
3.3.1 pH
The measured pH of the dry-hopped beers over 14 days of dry-
hopping is presented in figure 7. The results show, in agreement
with a previous publication [14], an increase in beer pH during
dry-hopping (Figure 7). Furthermore, our results show greater in-
crease in beer pH at lower dry-hopping temperatures (4 °C) than at
higher dry-hopping temperatures (19 °C); suggesting an impact of
dry-hopping temperature on this parameter. The pH increase was
observed for both hop varieties used in this study. This observation
is of course important for the perception of bitterness in beer, since
perceived bitterness intensity can be influenced by beer pH [14].
The increased beer pH may however be beneficial for the stability
of iso-α-acids during storage, since these compounds are less
prone to degradation at higher beer pH [9].
3.3.2 ABV (%)
A very interesting finding of this study relates to changes in beer
alcohol content during dry-hopping. Over the course of dry-
hopping, our results showed an increase in the alcohol content of
the dry-hopped beers (Figure 8). This increase was greater (for
both varieties) at the higher dry-hopping temperature investigated,
i.e. at 19 °C. Our study found an increase of approximately 7 %
in the alcohol content of beers dry-hopped at 19 °C. This finding
has significant implications for craft brewers not just in terms of
beer flavour, but also potentially in terms of their obligation to pre-
sent accurate information about the alcohol contents of products.
This sector has, in general, both a lower degree of control over
packaged beer % ABV and a propensity to utilise dry hopping at
very high dose rates.
3.3.3 Density
In addition to the observed increase in beer ABV (%), a conco-
Fig. 6 Increases in the total polyphenol content of beers dry-
hopped with (A) Hersbrucker and (B) Zeus for 14 days at 4
and 19 °C. Data point labels show the relative percentage
increase in total polyphenol content of beer during dry-
hopping
Fig. 7 pH of dry-hopped beers at 4 and 19 °C for (A) Zeus and,
(B) Hersbrucker
0
38
49 49 47 47
0
22
28 29 30
25
200
225
250
275
300
325
350
375
400
0 2 4 6 8 10 12 14
Total Polyphenol Content (mg/L)
Day
A. Hersbrucker
19°C
4°C
0
28
33 35 34 34
0
9
13 15 15 14
200
225
250
275
300
325
350
375
400
0 2 4 6 8 10 12 14
Total Polyphenol Content (mg/L)
Day
B. Zeus
19°C
4°C
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mitant reduction in beer density was observed over the course
of dry-hopping (Figure 9). This reduction in density and increase
in % ABV during dry-hopping was of greater magnitude at 19 °C,
compared to 4 °C, for both hop varieties. In each case, there was
a drop of approximately 0.5 % in beer density over the 14-day
dry-hopping period. These findings indicate further attenuation
of beer during the dry-hopping process, especially at higher
dry-hopping temperatures, and suggest that further fermentation
was initiated during the dry-hopping process. This may be due
to the rousing process re-initiating fermentation of any available
residual fermentable sugars in the beer, or the added dry hops
serving as an additional source of sugars for yeast metabolism.
Hops have been reported to contain approximately 2 % (w/w)
monosaccharides [2], which could potentially be extracted into
beer during dry-hopping (depending on alcohol content of beer
and hop addition rate) and utilised by yeast for the production of
more alcohol. Based on the monosaccharide content of hops,
dry-hopping at a rate of 20 g/L could potentially add an additional
400 mg of sugar per litre of beer during the dry-hopping process.
Recent reports have also suggested that this observation, ter-
med ‘hop-creep’ by some brewers may be due to the effect of
hop enzymes in breaking down non-fermentable sugars to fer-
mentable sugars for the yeast to metabolise during dry-hopping
[10]. However, further attenuation of beers during dry-hopping
as a result of wild yeast (from added dry hops) cannot be totally
discounted [5].
3.4 Changes in the hop acid profile of spent hops
3.4.1 Losses of iso-α-acids
The observed reduction in concentration of beer iso-α-acids ob-
served in this and other studies prompted us to investigate this
matter further. The losses of these acids in the latter stages of
beer production is mainly due to their adsorption onto the hops
used for dry-hopping [13]. The results of this study in this regard
are shown in figure 10 and confirmed that iso-α-acids are indeed
adsorbed onto spent hop materials which brewers dispose of after
dry-hopping. In figure 10A, the main iso-α-acid peaks separated
by HPLC are shown as a reference. In figure 10B, the hop acids
present in the Hersbrucker variety used for dry-hopping is pre-
sented, showing low levels of humulinones, α-acids and β-acids.
In figure 10C, the hop acid composition of the spent hop slurry is
presented, and immediately it can be observed (by simple retention
time comparison) that iso-α-acids are present in the recovered
spent hops. As shown in figure 10B, iso-α-acids were not present
in the fresh hops used for dry-hopping, but after the dry-hopping
process the spent hop slurry clearly illustrates the presence of
iso-α-acid compounds. Furthermore, on closer inspection of the
chromatogram of the spent hop, numerous oxidized hop acid
compounds which are likely to be highly polar in nature were
found to be present. It would be useful to identify some of these
compounds with a view to better understanding their potential
contribution to beer bitterness.
3.4.2 α-acids
The levels of α-acids remaining in the spent hops after dry-hopping
was a further area addressed in this study. We found that approxi-
Fig. 8 Measured alcohol content of dry-hopped beers at 4 and
19 °C for (A) Zeus and, (B) Hersbrucker
Fig. 9 Measured density of dry-hopped beers at 4 and 19 °C for
(A) Zeus and, (B) Hersbrucker
mately 15–25 % of the original -acid content remained in the spent
hop after 14 days of dry-hopping under the conditions investigated,
although this percentage can be expected to vary depending on
the specific dry hopping conditions (temperature, duration, α-acid
content, static/dynamic system etc.) adopted.
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Fig. 10 HPLC separation of hop acids in fresh and spent hops after 14 days of dry-hopping at 4 °C. (A) Reference peaks for iso-α-acids:
trans-isocohumulone (18.17), cis-isocohumulone (18.82), trans-isocohumulone (20.70), cis-isocohumulone & trans-isoadhumulone
(21.38), cis-isoadhumulone (22.24). (B) Hop acid profile of fresh Hersbrucker; humulinones: cohumulinone (13.26), humulinone
(14.96), adhumulinone (15.62); α-acids: cohumulone (23.42), prehumululone (24.77), humulone (27.07), postadhumulone (28.06) and
adhumulone (28.80); β-acids: colupulone (35.85), lupulone (39.00) and adlupulone (39.58). (C) Hop acid profile of spent Hersbrucker
hops, humulinones: cohumulinone (13.20), humulinone (14.95), adhumulinone (15.61); iso-α-acids: trans-isocohumulone (18.17),
cis-isocohumulone (18.83), trans-isocohumulone (20.71), cis-isocohumulone & trans-isoadhumulone (21.39), cis-isoadhumulone
(22.25); α-acids: cohumulone (23.40), prehumululone (24.72), humulone (27.04), postadhumulone (28.03) and adhumulone (28.78);
β-acids: colupulone (35.76), lupulone (38.91) and adlupulone (39.50)
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3.5 Changes in total polyphenol content of spent hops
The TPC of fresh and spent Hersbrucker hops as determined by
the Folin-Coilcateau assay is presented in table 1. The results
show an average TPC of 282 and 252 mg GAE/L for fresh Zeus
and Hersbrucker hop polyphenol extracts, respectively. In the
spent hops recovered after dry-hopping at 19 °C for 14 days, the
total polyphenol content remaining was 144 and 112 mg GAE/L
of hop extracts, respectively. At this temperature, these numbers
represent a reduction in TPC in the spent hops of 49 and 56 %
in the spent Zeus and Hersbrucker hops, respectively. After dry-
hopping at 4 °C for 14 days, the total polyphenol content of spent
Zeus and Hersbrucker was 205 and 162 mg GAE/L of hop extracts,
representing a reduction in total polyphenol content of 27 and
36 %, respectively. These lower values at the lower dry-hopping
temperature (4 °C) mean that less polyphenols were extracted
out of hops into beer at this temperature relative to 19 °C. This
further emphasises the significant effect of temperature on the
dynamic extraction of hop polyphenols and related compounds
into beer as discussed in Section 3.2. Greater reductions in TPC
of the spent hops were observed at 19 °C than at 4 °C for both
hops. Furthermore, these results also confirm greater extraction
of polyphenols into beer during dry-hopping from the Hersbrucker
hops than from Zeus, as was already identified from the beers
themselves in Section 3.2. Accordingly, approximately 50 % of
the original content of hop polyphenols remains in the spent hops
that brewers currently dispose of as waste. The polyphenol and
residual hop acid contents of spent hop slurries indicate that the
slurries could be an added-value material for brewers to consider,
for example, by re-introducing good quality spent hops back into
the kettle for part of the bittering of a fresh batch of wort.
4 Conclusions
This study investigated the impacts of dry-hopping at 4 and 19 °C,
using a low alpha (Hersbrucker) and high alpha (Zeus) hop vari-
ety, on the resulting profiles of several hop acid compounds over
a 14-day period. Our results suggest that the concentration of
bitter tasting humulinones in dry-hopped beers is not solely down
to the extraction of these compounds from hops during the dry-
hopping process, and that the formation of these compounds from
(α-acids) can occur during dry-hopping. Humulinone concentrations
during dry-hopping were significantly impacted by hop variety
and temperature; dry-hopping with high alpha hops at warmer
temperatures (19 °C) resulted in higher concentrations of these
Table 1 Total polyphenol content of fresh Hersbrucker and Zeus
hops and their respective spent materials after 14 days
of dynamic dry-hopping
Hop variety Hop
condition GAE (mg/L) % loss
Zeus Fresh 282 –
Hersbrucker Fresh 252 –
Zeus @ 19 °C Spent 143 49
Hersbrucker @ 19 °C Spent 11 2 56
Zeus @ 4 °C Spent 205 27
Hersbrucker @ 4 °C Spent 162 36
compounds compared to dry-hopping at 4 °C with a low alpha hop.
Humulinones are worthy of greater attention from craft brewers
who wish to better understand both the analytical and sensory
bitterness of their products, due to their bitterness intensity and
their high concentrations in dry-hopped beers.
We also provide conclusive evidence to show that losses of iso-
α-acids through dry hopping occurred due to their adsorption onto
spent hop materials. The greatest drop in the concentration of
iso-α-acids was observed after 24 h of dry-hopping at both tem-
peratures investigated. A significant increase in α-acid concentra-
tions during dry-hopping was only observed in beers dry-hopped
with high alpha Zeus at the higher dry-hopping temperature of 19
°C. The total polyphenol content of dry-hopped beers increased
significantly after just 24 h of dry-hopping for both hop varieties
and at both temperatures investigated; with the rate of polyphenol
extraction found to be significantly dependent on dry-hopping
temperature and hop variety. There was greater extraction of
polyphenols into beer at 19 °C than at 4 °C in both hop varieties,
although at both temperatures, more polyphenols were extracted
into the dry-hopped beer from Hersbrucker hop than from Zeus.
This is logical, as low alpha hop varieties usually contain more
polyphenols than high alpha hop varieties [17]. Significantly, dry-
hopping also had an impact on key beer analytical parameters.
In summary, we observed an increase in beer pH, a decrease in
beer density and increase in alcohol content over the course of
dry-hopping with both hop varieties. The effects on %ABV and
density were greater at the higher dry-hopping temperature of 19
°C; we conclude that the presence of suspended yeast in beer
during dry-hopping may well have initiated further fermentation
during this process – with sugars extracted from the dry hops
into beer during dry-hopping further promoting yeast metabolism.
Consequently, the presence of suspended yeast in beer during
dry-hopping may have further significant impacts on overall beer
flavour beyond those already observed in relation to volatile hop
compounds. Alternatively, our observation in relation to further
attenuation during dry-hopping may be due to the activity of
wild yeast (from the dry hops) introduced during dry-hopping
[5]. Brewers dry-hopping beer with some yeast in suspension
should endeavour to measure beer parameters (pH, ABV etc.)
after dry-hopping.
The results presented on spent hops provide some basis for brewers
to consider utilising spent hops in the brewhouse as a sustainable
approach towards reducing waste in the brewing process, and
as a potential solution to the shortage of hops created by high
demand. Dry hopping remains an exciting process which offers
brewers opportunities to experiment and stand out by creating
unique beers. Continued research into this topic will no doubt
further our knowledge of this complex, yet important process for
beer production.
Acknowledgement
The authors wish to acknowledge the financial support of SABMil-
ler plc and the University of Nottingham, as well as the technical
support provided by David Greening in the brewing trials conducted
for this study. Thanks to Anheuser-Busch InBev for permission to
publish this work.
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Received 22 August 2017, accepted 16 November 2017
