Journal of Food Composition and Analysis 106 (2022) 104278

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Influence of different fermentation conditions on the analytical and sensory

properties of craft beers: Hopping, fermentation temperature and
yeast strain
Remedios Castro a, Ana Belén Díaz b, Enrique Durán-Guerrero a, *, Cristina Lasanta b
a
Analytical Chemistry Department, Faculty of Sciences-University Institute of Wine and Food Research (IVAGRO-CAIV), University of Cadiz, Agrifood Campus of
International Excellence (CeiA3), Polígono Río San Pedro, s/n, Puerto Real, 11510, Cadiz, Spain
b
Chemical Engineering and Food Technology Department, Faculty of Sciences-University Institute of Wine and Food Research (IVAGRO-CAIV), University of Cadiz,
Agrifood Campus of International Excellence (CeiA3), Polígono Río San Pedro, s/n, Puerto Real, 11510, Cadiz, Spain

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

Keywords: The influence of the hopping method (boiling stage hopping or dry hopping), the fermentation temperature (12
Craft beers and 18 ◦ C), and the yeast strain (five different yeasts) on the physical-chemical characteristics (bitterness, color,
Dry hopping alcohol content), the phenolic content, the volatile compounds, as well as the sensory profile of beers, has been
Volatile compounds
studied. The hopping method was much more influential than temperature and yeast strain, however, its in­
Phenolic compounds
Sensory analysis
fluence on volatile content clearly depended on the fermentation temperature, with a higher content of volatile
compounds, when a higher fermentation temperature was employed. For phenolic compounds, dry hopping
produced less polyphenol-rich beers, regardless of the fermentation temperature. Sensorily, it resulted in more
floral and fruity beers than those produced by boiling stage hopping, which exhibited a more intense toasted
aroma. Concerning yeast strain, no relationship could be established between the type of commercial yeast
(bottom or top-fermenting) and the resulting volatile content.

1. Introduction to their antioxidant and antimicrobial capacity and also to their ability
to stabilize its foam.
Craft beers have gained popularity in recent years, and interest Hops are generally classified according to their concentration in
continues to grow due to the search for new and complex aromatic α-acids, thus, bitter hops contain more than 5% of these compounds,
profiles and unique aromas, such as malted barley, chestnuts or honey, while aromatic hops usually contain less than 5% and are characterized
among others (Gómez-Corona et al., 2016; Jaeger et al., 2020). by their aromatic profile (Palmer, 2006). However, there are hops
Hop (Humulus lupulus L., Cannabaceae family) is one of the raw characterized for providing beer with both bitterness and aromatic
materials of beer. It provides bitterness, taste and aroma (Machado et al., compounds. The main compounds responsible for the hops aroma are
2019a; Martins et al., 2018). It also contributes to the preservation of esters, which are associated with fruity characteristics; monoterpenes
beer. These characteristics are mainly due to the secondary metabolites (citric, spicy, resinous and herbaceous aromas); sesquiterpenes (spicy
that are present in the lupulin glands located in the female cones of the and wood aromas); ketones (floral notes); sulfur compounds (sulfur and
plant. Those metabolites can be fundamentally classified into resins, fruity notes); and aldehydes, which are associated with herbaceous
polyphenols and essential oils (Steenackers et al., 2015). The main resins aromas (Machado et al., 2019a; Martins et al., 2018).
in hops are divided into humulones (α-acids) and lupulones (β-acids), Hops can be added during wort boiling, in the whirlpool and during
and are responsible for the bitter taste of beer, while the characteristic or at the end of the fermentation. If hops are added during the boiling
aromas of the hops are provided by their essential oils (Machado et al., stage, the α-acids are isomerized to iso-α-acids, which are responsible for
2019b). The phenolic compounds in them contribute to the conservation the bitter taste of beer and which contribute to the stability of the foam
and stabilization of the organoleptic characteristics of beer, mainly due (Bland et al., 2015; Česlová et al., 2009). However, this method presents

* Corresponding author.
E-mail addresses: [email protected] (R. Castro), [email protected] (A.B. Díaz), [email protected] (E. Durán-Guerrero), cristina.lasanta@
uca.es (C. Lasanta).

https://doi.org/10.1016/j.jfca.2021.104278
Received 15 September 2021; Received in revised form 4 November 2021; Accepted 9 November 2021
Available online 16 November 2021
0889-1575/© 2021 Elsevier Inc. All rights reserved.
R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

Table 1
Yeast strains employed and means ± standard deviations according to the yeast employed for physical-chemical parameters.
Yeast
Parameter
1 2 3 4 5

Strain Us West Coast Ale Bohemian Lager Empire Ale New World Stronge Ale California lager
Strain Characteristics (according to Top-fermenting; can work Bottom- Top-fermenting; Top-fermenting Bottom-fermenting strain
manufacturer) in wide temperature and fermenting; fruity complex and fruity with the ability to ferment
sugar ranges; citric aroma and fresh aroma aroma at typical ale temperatures
Bitterness (ºIBU) 11.7±2.5 10.3±2.0 10.3±2.1 12.0±1.8 11.1±1.2
Alcoholic content (%, v/v) 4.98±0.25 b 4.99±0.20 b 4.73±0.23 c 5.25±0.28 a 4.98±0.27 b
Color (ºEBC) 23.4±6.8 23.3±5.8 22.4±5.1 23.1±6.4 22.4±6.5

Different letters in the same row indicate significant differences according to Tukey test (p < 0.05).

the inconvenience that most of the volatile compounds provided by the employed, Table 1). A 100 L batch of sweet wort was prepared using
hops may evaporate at high temperatures while other interesting com­ commercial dry malt extract (Spray dried malt extract light, Muntons,
pounds are degraded (Oberholster and Titus, 2016; Steenackers et al., UK; color EBC 10 % solution: 12 maximum; moisture: 5% maximum;
2015). If added during or at the end of this stage — late hop addition — protein: 5–7 % maximum; pH: 5–6). The wort was then separated into
the hops provide less bitterness, but they are mainly used for their two parts of 50 L each, one was used to produce the boiling stage hopped
contribution to the beer taste and aroma. In dry hopping methods, the beers (BS) and the second part was used to produce beer by dry hopping
hops are added during the fermentation or conditioning stages of the (DH).
beer (Oberholster and Titus, 2016; Wolfe, 2012). In those cases, For the BS method, 1.5 g of hops pellets of the Perle variety
non-isomerized α-acids are solubilized into the beer to contribute to the (Laguilhoat, Spain) were added per liter of wort before boiling at 100 ◦ C
taste of the beer in their oxidized forms, humulinones (Algazzali and for 90 min. The beers produced by DH were also boiled at 100 ◦ C for 90
Shellhammer, 2016). min but with only half the amount of the same hops variety added per
Dry hopping is a fairly simple technique used to intensify the aroma liter of wort (i.e. 0.75 g/L). Once the boiling stage had been completed,
and taste of beer. When dry hopping methods are used, hops are usually and after the subsequent centrifugation and washing with water, a
added in different forms (pellets, cones or hop essential oils) (Wolfe, 12.1◦ P wort was obtained, which was tempered at 15 ◦ C before entering
2012). The chemical process consists of a cold extraction of volatile and the fermentation stage.
non-volatile compounds that improves the aroma and microbial stability Five commercial beer yeast strains from Mangrove Jack’s (Auckland,
of the beer (Lafontaine and Shellhammer, 2018). Moreover, it is known New Zealand), all from the Saccharomyces cerevisiae species, were
that this technique allows the extraction of polyphenols and other employed for fermentation (Table 1). Three of them had been described
compounds that contribute to the bitterness of beer (Oladokun et al., by the manufacturer as top-fermenting yeasts (Y1, Y3, Y4), another one
2017a,b). as bottom-fermenting (Y2) and the last one was a bottom-fermenting
Different parameters can be modified during dry hopping, such as strain with the ability to ferment at typical ale temperatures (Y5). All
the static or agitated extraction of the hop compounds (Wolfe, 2012), of them were used for fermentation at two temperatures, 12 ◦ C and 18
different temperatures, presence or absence of yeast, varying amounts of ◦
C, and this resulted in 20 different types of beer when the two hopping
hops, etc. (Lafontaine and Shellhammer, 2018). A new trend in craft options are considered. Each production process was also carried out in
brewing consists of adding a larger amount of hops, using different va­ duplicate. The yeasts were previously rehydrated in warm water and
rieties, and developing new recipes and techniques to enhance the then dosed at 10 g/23 L wort, as recommended by the manufacturer.
extraction of the aromatic compounds of interest, as well as the com­ The fermentation process was carried out using 2 L glass flasks, with
pounds responsible for the bitter taste of beer (Eyres et al., 2007). 1.8 L of wort in each flask. Two temperature-controlled cabinets were set
Together with hops, fermentation temperature and yeast strain can at 12 and 18 ◦ C for each fermentation batch. The first fermentation was
have a significant impact on the physical-chemical and sensory char­ continued controlled until a constant density measurement (Plato de­
acteristics of beer (Webersinke et al., 2018). In a previous study, the gree) below 3.5◦ P was obtained for three consecutive days. This
influence of both the type of yeast used and the fermentation tempera­ fermentation took ca 3 weeks at 12 ◦ C and 2 weeks at 18 ◦ C. After the
ture on the phenolic and volatile content, as well as on the sensory first fermentation, the content in each fermenter was separated into two
characteristics, of craft beers was investigated (Lasanta et al., 2020). 1 L bottles. According to the fermentation temperature, the amount of
However, no work has been found in the literature on the influence of dissolved CO2 would be 2.18 g/L of fermenter at 12 ◦ C and 1.80 g CO2/L
the hopping method on the physical-chemical and sensory profile of of fermenter at 18 ◦ C. An amount of sugar between 6 and 8 g/L was
craft beer when different yeast strains and fermentation temperatures added to each bottle to reach a standard amount of 2.6 g of CO2/L of
are used. fermenter after the second fermentation. In the case of the beers pro­
Therefore, the main objective of this work is to determine the relative duced by the DH method, another 0.75 g/L of the same hops used pre­
influence of the hopping method, the fermentation temperature and the viously were added to the fermenter to equal the total dose of hops in
yeast strain on the physical-chemical characteristics (bitterness, color, beers produced by BS (1.5 g/L altogether). To control the second
alcoholic strength), phenolic content, volatile compounds, as well as fermentation, the turbidity of the bottle was observed with the help of a
sensory profile, of beers. light source, until every particle had sedimented onto the bottom of the
bottle and the beer was perfectly clear. The second fermentation and
2. Materials and methods clarification stage took 5 weeks at 12 ◦ C and 4 weeks at 18 ◦ C. After this,
all the beers were matured for 2 weeks at 15 ◦ C, for the stabilization of
2.1. Beer making procedures aroma and taste. Measurements to determine the physical-chemical and
sensory characteristics, the phenolic content and the volatile compounds
All the beers in our study were brewed following the same meth­ of the different beers were performed after maturation.
odology, with the main change of the phase in which the hop is added
(boiling stage hopping and dry hopping); the fermentation temperature
(12 and 18 ◦ C); and the yeast strain (five different strains were

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R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

2.2. Determining physical-chemical parameters 2.5. Sensory analysis

The degrees plato (ºP) were determined using a 0–10 scale densim­ A panel of 10 tasters (6 women and 4 men), all between the ages of
eter calibrated at 20 ◦ C (Alla France, France). The alcoholic strength and 30 and 62, was appointed to carry out the sensory evaluations. All of
color (ºEBC), were determined using the official analytical method them had extensive previous experience in the sensory evaluation of
established by the Analytical Division of the European Brewery different foods, and 4 of them were members of official tasting panels (P.
Convention using an Anton Paar densimeter model DMA 500 and a D.O. Sherry Wine and P.D.O. Sherry Vinegar). In this work, additional
Genesys 10 UV spectrophotometer, respectively. The bitterness units training in beer samples was provided once a week for 3 months. During
were determined according to ASBC methodBeer-23A (American Soci­ the first phase of the training, the judges selected five aromatic attri­
ety of Brewing Chemists, 2011). butes: cereal, herbaceous, fruity, floral and toasted. During the second
part of the training, the judges quantified the intensity of each attribute
2.3. Analysis of volatile compounds in different samples (30 commercial craft beers) in order to homogenize
their criteria.
Volatile compounds were identified and quantified by gas chroma­ The tested samples were presented at a controlled temperature of 20
tography with detection by mass spectrometry (GC–MS), after extraction ◦
C in glass cups. The tasting sessions were carried out in a standardized
using Stir Bar Sorptive Extraction (SBSE) (Ruvalcaba et al., 2019). tasting room (ISO, 2007a), where the samples were presented in random
Briefly, the SBSE methodology applied included: 50 mL of beer; 12.5 g of order and coded following a three-digit code (ISO, 2017b).
NaCl (25 %, v/w); polydimethylsiloxane stir bars (PDMS; 1 mm x 0.5 The sensory analysis was carried out in four sessions involving 10
mm) (Gerstel, Mülheim a/d Ruhr, Germany); and agitation at 1000 rpm different beer samples in each session. A descriptive aroma tasting was
for 3 h. For the desorption, a commercial TDS-2 thermal desorption unit carried out, and additionally the tasters were asked to fill in a "global
(Gerstel) connected to a programmed-temperature vaporization (PTV) evaluation" section in which they performed a hedonic evaluation of
injector CIS-4 (Gerstel) by a heated transfer line was used. This system each product according to a 6-point scale. A tasting card including the
was connected to an Agilent 6890 GC (Agilent Technologies, Palo Alto, five selected attributes was filled in according to a numeric scale from
CA, USA) equipped with a DB-Wax capillary column (J&W Scientific, 0 to 5, where 0 meant absence and 5 very high intensity.
Folsom, CA, USA), 60 m × 0.25 mm I.D., with a 0.25 μm coating. The
carrier gas used was helium (1.0 mL/min). The oven temperature was
2.6. Statistical analysis
programmed at 35 ◦ C for 10 min; ramped at 5 ◦ C/min up to 100 ◦ C;
ramped at 3 ◦ C/min to 210 ◦ C; and held for 40 min. The mass detector
Several statistical tools were employed for data analysis: Analysis of
was an Agilent 5973 MS (Agilent Technologies) operated in EI+ mode at
Variance (ANOVA), Multiple Analysis of Variance (MANOVA), Principal
70 eV in a 30–400 amu range. All the samples were analyzed in
Component Analysis (PCA) and Cluster Analysis (CA). All of them were
duplicate.
carried out using Statgraphics Centurion XVII (Statpoint Technology
For the correct identification of all the volatile compounds, the linear
Inc., Virginia, USA) and Statistica 12.5 (StatSoft, Inc., Tulsa, USA). The
retention indices were calculated employing a C6–C30 n-alkane series.
data were standardized ((value-mean)/standard deviation) and Tukey’s
The mass spectra library Wiley (Wiley 7 N Edition Library, 7th Edition,
test was applied to data pairs with a significant difference (p < 0.05).
2000) was also employed. The quantification was performed following
the external standardization method. The calibration curves corre­
sponding to 6 concentration levels, in duplicate, prepared in a synthetic 3. Results and discussion
beer matrix (5% ethanol–water mixture) were elaborated for each
compound using commercial standards. All of them presented purity 3.1. Physical-chemical parameters
levels above 99 % and were acquired from Sigma-Aldrich (St. Louis, MO,
USA). The average values obtained for alcoholic strength, color and
bitterness, depending on the yeast, temperature and hopping method
2.4. Analysis of phenolic and furanic compounds used for the different beers produced are shown in Tables 1 and 2.
The variety of yeast strain did not significantly influence the bitter­
All the data were acquired and processed by Empower 1 (Waters ness or color of the beers. Regarding the alcoholic degree, slight dif­
Corporation, Milford, MA USA). A Water Acquity UPLC system (Waters ferences can be observed. Yeast Y4 provided a slightly higher alcoholic
Corporation) equipped with a diode array detector (DAD) was used for degree than the other yeast strains, while the beer elaborated with yeast
the determination, in duplicate, of polyphenols and furanic compounds Y3 reached the lowest alcoholic degree (Table 1). However, when we
using the conditions previously optimized by Schwarz et al. (2009). An look at the samples according to the temperature and the hopping
Acquity UPLC BEH C18 column (100 × 2.1 mm i.d., with 1.7 μm particle method used (Table 2), significant differences can be observed in terms
size), also from Waters, was used. The column temperature was main­ of bitterness. Thus, the beers elaborated at a lower temperature, and
tained at 47 ◦ C. The binary system phases were: A (3% ACN, 2% acetic those obtained through the traditional hopping method presented
acid, 95 % water) and B (85 % ACN, 2% acetic acid, 13 % water), with a higher ºIBU. These results are consistent with previous studies, where an
flow rate of 0.7 mL/min. The injection volume was 2.5 μL. The 6.5 min
gradient was as follows: 0 min, 100 % A, 3 min, 90 % A (curve 6), 4 min, Table 2
90 % A, 6.5 min, 25 % A (curve 6). Finally, the column was washed with Means ± standard deviations for the physical-chemical parameters according to
100 % B for 3 min and equilibrated with 100 % A for 3 min. All the type of hopping and fermentation temperature.
samples were filtered through 0.22 μm nylon filters from Scharlab Temperature Type of Hopping
Parameter
(Barcelona, Spain). For identification and quantification purposes, 18 ºC 12 ºC BS DH
different commercial standards purchased from Fluka (Buchs,
Bitterness (ºIBU) 9.59±1.98 12.5±1.2 a 11.7±2.1 a 10.4±1.8 b
Switzerland) and Sigma were employed. For the identification of the b
different polyphenols and furanic compounds, both their retention times Alcoholic content (%, 4.92±0.26 5.05±0.32 5.00±0.23 4.97±0.35
and UV–vis spectra were considered. The external standardization v/v)
method was used for the quantification. Benzoic acids and furans were Color (ºEBC) 23.1±6.3 22.8±5.7 28.8±1.4 a 17.1±0.8 b

quantified at 280 nm, whereas hydroxycinnamic acid derivatives were For each parameter (temperature and type of hopping) different letters in the
measured at 320 nm. same row indicate significant differences according to Tukey test (p < 0.05).

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R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

inverse function was observed between the fermentation and/or matu­ analyzed by multivariate variance, and yeast, fermentation, tempera­
ration temperature of the beer and its degree of bitterness (Lasanta et al., ture, and hopping method were considered as possible influential fac­
2020; Nimubona et al., 2013). It is also known that the α-acids contained tors, it was observed that, according to our expectations, the three
in the hops are isomerized into more bitter compounds (iso-α-acids) at factors exerted important influences (p < 0.05) on the volatile content of
boiling temperatures (Haseleu et al., 2010; Hough et al., 1982). It is, the resulting beers (Table 3). The beers that had been fermented at 18 ◦ C
therefore, usual in beers where all the hops have been added according and to which the DH technique had been applied had a higher content of
to the BS method, i.e. at the brewing stage, to exhibit a higher level of volatile compounds (Table 1S, supplementary material). Similar results
bitterness than those obtained through DH (where only a part of the had been previously observed by other authors (Haslbeck et al., 2018a,
hops are added during the brewing stage). However, this difference was b), with higher content of terpenols and esters in those beers in which
not as wide as one might expect, since DH produces other types of hops were added at a later stage in the brewing process
compounds, such as humulinones, which also contribute to the bitter­ Other authors (Dresel et al., 2015) reported significant differences
ness of the final beer, regardless of the temperature used (Oladokun between the volatile profile of the wort and that of the beer, with a
et al., 2017a,b). The hop variety employed (Perle) is characterized by significant increase in volatile content after the fermentation process.
being mostly used to brew traditional beer types like Pilsen or Lager and For these authors, the amounts determined for most of the volatile
is generally used during wort boiling due to its bittering properties, not compounds were greater in the dry hopped beers, with the concentra­
to produce dry-hopped beers. However, in this work, in order to tion levels of saturated esters and higher alcohols mainly dependent on
compare its influence on analytical and sensory properties with those the fermentation process and not on the hop variety used.
from other factors such as fermentation temperature and yeast strain, In the present study, as far as fermentation strains are concerned Y2
the same hop variety was employed for both hopping methods. and Y3 produced beers with higher content of volatile compounds, while
As for the color, the BS beers presented significantly higher values Y1 produced beers with significantly lower content of the same (Table 3
than the DH beers. This fact was later confirmed by the results registered and Table 2S, supplementary material). It should be reminded that
for phenolic content. strains Y1, Y3 and Y4 had been commercially described as top-
When the physical-chemical data were subjected to multivariate fermenting yeasts whereas Y2 was a bottom-fermenting one. It seems,
statistical analysis of the clusters (Fig. 1), the dendrogram obtained therefore, that no relationship can be established between type of
allowed two great clusters to be observed: one corresponding to the DH commercial yeast and the resulting volatile content and that each yeast
samples and another one including the BS samples. Within these clus­ strain can produce a specific volatile profile in beer, as observed by other
ters, the samples were divided into two subclusters with each hopping authors (Haslbeck et al., 2018a).
method, according to the fermentation temperature. Therefore, ac­ An increase in the fermentation temperature provoked an increase in
cording to physical-chemical parameters, the hopping method was the the concentration of the majority of the volatile compounds studied
most influential factor, followed by the temperature. (Table 1S, supplementary material). This is consistent with previous
studies, where it was observed that the concentration of volatile com­
pounds in general, and acetaldehyde, higher alcohols and esters, in
3.2. Volatile compounds particular, increased in all the beers as the temperature was increased
(Šmogrovičová and Dömény, 1999; Webersinke et al., 2018).
A total of 41 volatile compounds from the ester, aldehyde, alcohol, The multivariate analysis also revealed significant variations of
acid, furan, terpene, and ketone groups were detected in the different volatile content levels caused by the interaction between these factors.
beers in the study (Table 1S and Table 2S in the supplementary Particularly, the "hopping method-temperature" interaction, in general
material). terms, showed a greater positive influence from the temperature factor
The group of esters included acetates and ethyl esters. Among the on those beers produced through the DH method than on those beers
former, ethyl acetate is the one that usually appears at higher concen­ produced by BS (Table 3).
trations in beers and represents approximately one-third of all the esters. As far as strain-temperature interaction is concerned, the beers fer­
From this ester group, the “banana” and “pineapple” aromas provided mented using Y2 (commercially classified as bottom fermenting), Y3
by isopentyl acetate and ethyl pentanoate were detected in beers when (top fermenting) and Y4 (top fermenting) strains exhibited a higher
concentrations were higher than 2 ppm and 0.1 ppb, respectively volatile content when the fermentation temperature was increased (data
(Šmogrovičová and Dömény, 1999; Takeoka et al., 1998). not shown), while the Y5-fermented beers (top fermenting) did not seem
In our study, when the data obtained on volatile compounds were to be influenced by this factor. It can be inferred, as previously
mentioned, there was no clear correlation between the commercial
classification of the yeast strains used and the volatile profile of the beers
(Lasanta et al., 2020). Finally, in relation to the hopping method-strain
type interaction, it should be mentioned that no overall association
between hopping method and yeast strain was observed.
From a multivariate perspective, the data on volatile compounds
were subjected to Principal Component Analysis (PCA, Kaiser’s criterion
(eigenvalue >1)). Eight PCs were obtained that explained 83.3 % of the
actual variability between the samples. The PC1 explained 28.4 % of this
variability, while the PC2 explained 24.9 %. 1-Hexanol, 2(5 H)-
furanone, 3-methyl-1-butanol, ethyl heptanoate, alpha-terpinolene,
isovaleric acid, ethyl dodecanoate, ethyl benzopropanoate, beta-
phenylethyl-2-methylbutyrate, 1-decanol, and octanol, all with posi­
tive values, were the volatile compounds with the highest weight in the
PC1. For the PC2, these were: 2-phenylethyl alcohol, phenylethyl ace­
tate, octanoic acid, octanal, nonanoic acid, acetic acid, and ethyl octa­
noate, all of them also with positive effects.
Fig. 1. Cluster Analysis of the beer samples based on the physical-chemical When the distribution of all the samples over the plane defined by the
parameters. Codification: Y: Yeast; 12/18: temperature of fermentation; BS: first two PCs is observed (Fig. 2), it can be noted that the DH samples
boiling stage hopping; DH: dry hopping. fermented at 18 ◦ C are grouped in the quadrant of the positive values of

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R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

Table 3
Multivariate analysis of variance on volatile content for yeast, temperature and type of hopping.
Compound Yeast (A) Temp. (B) Hopping (C) AB AC BC

ethyl acetate 0.0004* 0.0036* 0.0001* 0.0001* 0.0002* 0.0018*

isobutyl acetate 0.0002* 0.0000* 0.4593 0.0000* 0.0001* 0.5861
ethyl butyrate 0.0015* 0.8001 0.0000* 0.0348* 0.1607 0.0709
ethyl isovalerate 0.0694 0.0087* 0.0000* 0.0056* 0.0772 0.0945
hexanal 0.4051 0.1603 0.5188 0.3410 0.5718 0.3334
ethylpentanoate 0.0001* 0.0005* 0.0000* 0.0002* 0.0000* 0.0125*
isopenty acetate 0.0000* 0.0000* 0.0000* 0.0000* 0.0000* 0.0001*
3-methyl -1-butanol 0.0054* 0.0000* 0.0000* 0.0673 0.0048* 0.0000*
ethyl hexanoate 0.0000* 0.0000* 0.1552 0.0005* 0.0015* 0.7954
hexyl acetate 0.6243 0.0487* 0.0050* 0.1768 0.0185* 0.6267
octanal 0.1358 0.0051* 0.0000* 0.1409 0.5321 0.1265
ethyl heptanoate 0.7320 0.0000* 0.0000* 0.0155* 0.1023 0.0090*
1-hexanol 0.0002* 0.0000* 0.0000* 0.0005* 0.0108* 0.2867
heptyl acetate 0.0065* 0.0000* 0.0000* 0.0150* 0.0002* 0.0000*
nonanal 0.2417 0.8023 0.8031 0.3419 0.1949 0.0897
acetic acid 0.1854 0.4903 0.0000* 0.0071* 0.0824 0.1697
ethyl octanoate 0.0000* 0.0000* 0.1508 0.0109* 0.0036* 0.1521
heptanol 0.0003* 0.0000* 0.9431 0.6612 0.0004* 0.0553
octyl acetate 0.0883 0.0000* 0.0000* 0.0004* 0.0034* 0.0000*
benzaldehyde 0.0000* 0.0000* 0.8392 0.0000* 0.0267* 0.3858
alpha-terpinolene 0.5929 0.0003* 0.0000* 0.3018 0.3636 0.0005*
isobutyric acid 0.0017* 0.2563 0.0000* 0.0006* 0.0339* 0.0872
octanol 0.0000* 0.0000* 0.0000* 0.0000* 0.1074 0.0003*
2,3-dihydrobenzofurane 0.0030* 0.0023* 0.8349 0.0074* 0.0001* 0.0550
2-furanmethanol 0.0001* 0.0000* 0.0000* 0.0057* 0.0000* 0.0000*
ethyl decanoate 0.0000* 0.0000* 0.0067* 0.0000* 0.0974 0.9286
hexanoic acid 0.0000* 0.0000* 0.0751 0.0000* 0.1232 0.9668
isovaleric acid 0.0000* 0.0000* 0.0000 0.0001* 0.0000* 0.0001*
Ethyl benzenoate 0.0001* 0.0000* 0.0000* 0.0001* 0.0042* 0.0000*
2(5 H)-furanone 0.0084* 0.0001* 0.0000* 0.0083* 0.0332* 0.0007*
1-decanol 0.0000* 0.0000* 0.0000* 0.0000* 0.0003* 0.0099*
phenylethyl acetate 0.0063* 0.0000* 0.0000* 0.0056* 0.0001* 0.0000*
beta-damascenone 0.0396* 0.0335* 0.0000* 0.0037* 0.0007* 0.1367
ethyl dodecanoate 0.0000* 0.0000* 0.0000* 0.0001* 0.0101* 0.0215*
Ethyl benzopropanoate 0.0000* 0.0000* 0.0000* 0.0000* 0.9162 0.0010*
phenylethyl alcohol 0.0000* 0.0000* 0.0023* 0.0699 0.0002* 0.7055
octanoic acid 0.0000* 0.0000* 0.0134* 0.0918 0.0080* 0.8707
nonanoic acid 0.0000* 0.0000* 0.3191 0.0000* 0.0002* 0.1349
β-phenylethyl-2-methylbutyrate 0.0534 0.0001* 0.0000* 0.0008* 0.0028* 0.0360*
2-metoxi-4-vinylphenol 0.0000* 0.2306 0.0000* 0.0216* 0.0222* 0.0232*
decanoic acid 0.0000* 0.0000* 0.1514 0.0000* 0.0001* 0.1181
*
Values are significant at p < 0.05.

the PC1 and the negative values of the PC2, while those fermented at 12 and yeast strain (Table 4), as well as by their dual interactions. Of the
◦
C, whether DH or BS, are located in the quadrant corresponding to the three factors considered, the hopping method appears to play a signifi­
negative values of both PCs. These facts demonstrate a clear connection cant role with regard to a greater number of phenolic compounds. Thus,
between the hopping method and the fermentation temperature, which in general, a higher concentration of polyphenols was observed in those
results in greater volatile compounds content, especially in those vola­ beers elaborated through BS (Table 2). Gallic acid, chlorogenic acid, and
tile compounds with a positive contribution to PC1 mentioned previ­ ferulic acid presented higher concentration levels in beers that had been
ously, when the DH method is combined with fermentation at 18 ◦ C. fermented at 18 ◦ C, while tyrosol, vanillic acid and i-ferulic acid were
These facts were again confirmed by an analysis of the clusters found at higher concentrations in beers that had been fermented at 12
formed by the values associated with volatile compounds. A dendrogram ◦
C. With regard to the yeast strain used, Y1 and Y2 (top fermenting for
was drawn (Euclidean distance and Ward’s rule) as shown in Fig. 3. It the former and bottom fermenting for the latter) resulted in a higher
can be seen that three clusters were obtained: one corresponding to DH phenolic content, while Y3 and Y4 (both top fermenting) produced the
samples fermented at 12 ◦ C; another one corresponding to DH samples lowest phenolic content (Table 4S, supplementary material).
fermented at 18 ◦ C and an intermediate cluster for all the BS samples According to previous studies, the phenolic content of beer, both
fermented at either 12 or 18 ◦ C. It appears that, as already indicated by qualitatively and quantitatively, depends on the raw material (hops and
the previous results, the influence of DH on the volatile compound malted barley) as well as on the brewing process (Wannenmacher et al.,
content was dependent on the fermentation temperature. It is worth 2018). Subsequently, the fermentation process, warm rest and
mentioning that the hop variety that was employed is considered low- chill-lagering results in substantial reductions of its phenolic content.
aromatic, and it could be responsible for this dependence on fermenta­ Different studies on the DH effect at 4 and 19 ◦ C, revealed a higher
tion temperature in order to obtain beers with more volatile compounds extraction of polyphenols in the case of beers fermented at 19 ◦ C,
content when this hop variety is used in dry hopped beers. although extractions would vary depending on the variety of hops used
(Oladokun et al., 2017a,b).
3.3. Phenolic and furanic compounds Other authors (Ganbaatar et al., 2015) determined the evolution of
the phenolic content of different beers during their production process
As we could already observe in the case of volatile compounds, and reported uneven content variations of each separate polyphenol,
phenolic and furanic compounds were significantly affected by the three where some of them would decrease after boiling the wort together with
factors that have been considered in this study, i.e., hops, temperature the hops or over the fermentation process, others would remain constant

5
R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

composition.
The three factors considered in this work showed significant in­
teractions with respect to the phenolic content of the samples, as
observed in the case of their volatile content. These interactions were
more evident after studying the data on polyphenols and furanic com­
pounds obtained from the PCA (Fig. 4). In this analysis, 5 PCs were
obtained that explained 73.9 % of the variability between the samples.
The PC1, with 36.9 % of the variability, presented a high weight of
compounds such as 5-hydroxymethylfurfural and caffeic acid, while the
PC2, with 13.1 % of the variability, was mainly formed by tyrosol, and
vanillic and i-ferulic acids, with a positive value and gallic acid, with a
negative one. The PC1 separated DH beers from BS beers, having
negative values for the former and positive values for the latter. Like­
wise, great similarity in polyphenols was observed in DH beers regard­
less of the fermentation temperature. The PC2 separated the beers
produced by BS at different temperatures. Thus, the beers that were
fermented at 18 ◦ C exhibited negative values, while those fermented at
12 ◦ C presented positive ones. It seems therefore that the temperature
factor exerted a certain influence on the phenolic content of only those
beers that had been produced by BS, while it had no relevant effect on
the beers obtained through DH. These remarks were confirmed by
cluster analysis (Fig. 5), where two main groups could be clearly
observed: one formed by the DH beers and another one comprising the
BS beers, which were clearly affected by the temperature factor.
It seems then that dry hopping produced less polyphenol-rich beers,
regardless of the fermentation temperature, while the same beers were
richer in volatile compounds when fermented at 18 ◦ C.

3.4. Sensory analysis

Previous studies have determined the effect of the different yeast

Fig. 2. Principal Component Analysis of the beer samples based on the volatile strains on a number of fermented products and, naturally, they have also
composition. A.: Complete graph; B.: Zoomed region. Codification: Y: Yeast; 12/
examined how that diversity of strains may affect beer sensory profile
18: temperature of fermentation; BS: boiling stage hopping; DH: dry hopping.
(Larroque et al., 2021; Lasanta et al., 2020). In our study, in order to
separate such effect from the rest of the parameters, and taking into
account the previous analytical results in volatile and phenolic com­
pounds, four groups have been set up according to hopping method (BS
and DH) and fermentation temperature (12 and 18 ◦ C), each one being
the average of the different yeasts used.
Fig. 6 shows the mean values in each group for the 6 descriptors
evaluated by the judges. It can be observed that the aromatic profile was
clearly influenced by the hopping method used and thus higher values of
the cereal and toasted descriptors were observed for beers obtained by
BS, while higher scores for the herbaceous, fruity and floral descriptors
were assigned to the beers obtained by DH, the latter presenting the
most significant differences (ANOVA; p < 0.05). This increase in her­
baceous, fruity and floral descriptors in dry hopped beers might be the
reason for a greater perception of cereal and toasted descriptors in BS
beers in which the former notes were present in a lower proportion.
By adding part of the hops at the second fermentation stage, at a
much lower temperature than that required for the BS method (boiling),
Fig. 3. Cluster Analysis of the beer samples based on the volatile composition. we can prevent the evaporation of the volatile compounds that are
present in the hops, as well as their conversion because of the high
or even increased their concentration levels. Zhao (2015) established temperatures and the transformations that take place during the
that the polyphenol content in beers increases during malting and fermentation caused by the yeasts (Forster and Gahr, 2013; Steenackers
mashing, but decreases significantly during the subsequent fermentation et al., 2015). This general increase in volatile contents are consistent
and storage stages. This is probably explained by the precipitation and with the results from other authors, although most of them agree that
adsorption to yeast during the fermentation process. In our particular the specific effect depends on many other factors, such as the exact
case, the addition of part of the hops during the second fermentation moment of addition, the variety of hops, or even on their state of
(DH) seemed to lead to a lower extraction of phenolic substances from ripening (Dresel et al., 2015; Forster and Gahr, 2013; Lafontaine et al.,
the hops and thus a lower final phenolic content in the beers. The 2019; Sanz et al., 2019; Schnaitter et al., 2016).
possible influence of the composition of the hops, with a higher amount Furthermore, within each technique (BS or DH), by increasing the
in those BS beers, on the subsequent work of the yeasts during the fermentation temperature, higher values were generally obtained for all
fermentation process should also be taken into account in order to the descriptors studied, which can be explained by a greater activation
explain the differences observed for BS and DH beer polyphenolic of the yeasts and a greater production of the aromatic compounds
derived from the fermentation. From the overall evaluation, it can be

6
R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

Table 4
Multivariate analysis of variance on phenolic and furanic content for yeast, temperature and type of hopping.
Compound Yeast (A) Temp. (B) Hopping (C) AB AC BC

gallic acid 0.0000* 0.0000* 0.0012* 0.0012* 0.1202 0.0000*

5-hydroxymethyl furfural 0.0088* 0.1111 0.0000* 0.0822 0.2197 0.6752
furfural 0.0542 0.2848 0.0000* 0.3158 0.0791 0.2735
protocatechuic acid 0.0114* 0.6946 0.0000* 0.0006* 0.7297 0.0061*
tyrosol 0.0005* 0.0000* 0.0000* 0.0088* 0.0026* 0.0000*
vanillic acid 0.0116* 0.0002* 0.0031* 0.0051* 0.0072* 0.0000*
chlorogenic acid 0.0000* 0.0076* 0.0000* 0.0000* 0.0000* 0.1064
caffeic acid 0.0045* 0.1744 0.0000* 0.0001* 0.0000* 0.6823
p-coumaric acid 0.0000* 0.0755 0.0000* 0.0001* 0.0000* 0.0015*
ferulic acid 0.1119 0.0001* 0.0000* 0.3455 0.0785 0.0011*
i-ferulic acid 0.0000* 0.0000* 0.4019 0.0000* 0.0000* 0.0000*
*
Values are significant at p < 0.05.

Fig. 4. Principal Component Analysis of the beer samples based on the

phenolic and furanic compounds. Codification: Y: Yeast; 12/18: temperature of
fermentation; BS: boiling stage hopping; DH: dry hopping.

Fig. 6. Descriptive analysis of beers submitted to boiling stage hopping (BS)

and dry hopping (DH) at 12 ◦ C or 18 ◦ C of fermentation. Codification: 12 ◦ C/
BS ; 18 ◦ C/BS ; 12 ◦ C/DH ; 18 ◦ C/DH .

Fig. 5. Principal Component Analysis of the beer samples based on the

phenolic and furanic compounds.

concluded that the judges preferred beers brewed at 18 ◦ C and, in

particular, those brewed using the DH method (Fig. 6). This would be in
line with the results corresponding to volatile compounds, with higher
contents of alcohols and esters, which, according to previous literature
(Brendel et al., 2019; Haslbeck et al., 2018a), deliver an increase in
aromas in general, especially in floral and fruity aromas (Takeoka et al.,
1998), to beers brewed through DH and fermented at higher tempera­ Fig. 7. Principal Component Analysis of the beer samples based on the
phenolic and furanic compounds. Codification: Y: Yeast; 12/18: temperature of
tures (Tables 3 and 1S, supplementary material).
fermentation; BS: boiling stage hopping; DH: dry hopping.
For a better understanding of the results, the data from the principal
component analysis applied to the descriptors evaluated for the different
samples are shown in Fig. 7. The different beers obtained are repre­ observed how the PC1, mainly associated in a positive way to fruity and
sented on the plane defined by the first two main components together floral descriptors, and in a negative way to cereal and toasted, is the one
with the aromatic descriptors that were considered. These two compo­ that separates nearly all the samples elaborated by BS (with negative
nents explained 67.91 % of the total variance of the data. It can be values) from those elaborated by DH (with positive values). The PC2,
which is primarily related to the herbaceous descriptor and, to a lesser

7
R. Castro et al. Journal of Food Composition and Analysis 106 (2022) 104278

extent, to cereal, does not differentiate the samples so clearly, even Gómez-Corona, C., Escalona-Buendía, H.B., García, M., Chollet, S., Valentin, D., 2016.
Craft vs. industrial: habits, attitudes and motivations towards beer consumption in
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volatile and phenolic compounds content when compared to the lesser Shellhammer, T., 2019. Impact of harvest maturity on the aroma characteristics and
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strain, however, its influence, on some parameters depended on the Larroque, M.N., Carrau, F., Fariña, L., Boido, E., Dellacassa, E., Medina, K., 2021. Effect
of Saccharomyces and non-Saccharomyces native yeasts on beer aroma compounds.
fermentation temperature. Int. J. Food Microbiol. 337, 108953.
Lasanta, C., Durán-Guerrero, E., Díaz, A.B., Castro, R., 2020. Influence of fermentation
CRediT authorship contribution statement temperature and yeast type on the chemical and sensory profile of handcrafted beers.
J. Sci. Food Agric. 101, 1174–1181.
Machado, J.C., Faria, M.A., Ferreira, I.M.P.L.V.O, 2019a. Hops: New perspectives for an
Remedios Castro: Writing - original draft, Data curation, Software, Old beer ingredient. Natural Beverages. Elsevier, pp. 267–301.
Funding acquisition. Ana Belén Díaz: Investigation, Visualization, Machado, J.C., Faria, M.A., Melo, A., Martins, Z.E., Ferreira, I.M.P.L.V.O, 2019b.
Modeling of α-acids and xanthohumol extraction in dry-hopped beers. Food Chem.
Formal analysis. Enrique Durán-Guerrero: Conceptualization, Writing 278, 216–222.
- review & editing, Validation. Cristina Lasanta: Resources, Method­ Martins, C., Brandão, T., Almeida, A., Rocha, S.M., 2018. Unveiling the lager beer
ology, Project administration, Supervision. volatile terpenic compounds. Food Res. Int. 114, 199–207.
Nimubona, D., Blanco, C.A., Caballero, I., Rojas, A., Andrés-Iglesias, C., 2013. An
approximate shelf life prediction of elaborated lager beer in terms of degradation of
Declaration of Competing Interest its iso-α-acids. J. Food Eng. 116, 138–143 h.
Oberholster, A., Titus, B.M., 2016. Review: impact of dry hopping on beer flavor
stability. Ann Food Process Preserv 1, 1–6.
The authors declare that they have no known competing financial
Oladokun, Olayide, James, S., Cowley, T., Dehrmann, F., Smart, K., Hort, J., Cook, D.,
interests or personal relationships that could have appeared to influence 2017a. Perceived bitterness character of beer in relation to hop variety and the
the work reported in this paper. impact of hop aroma. Food Chem. 230, 215–224.
Oladokun, O., James, S., Cowley, T., Smart, K., Hort, J., Cook, D., 2017b. Dry-Hopping :
the effects of temperature and hop variety on the bittering profiles and properties of
Appendix A. Supplementary data resultant beers. Brew. Sci. 70, 187–196.
Palmer, J., 2006. How to Brew. Everything You Need to Know to Brew Beer Right the
Supplementary material related to this article can be found, in the First Time. Brewers Publications, Colorado (USA).
Ruvalcaba, J.E., Durán-Guerrero, E., Barroso, C.G., Castro, R., 2019. Development of a
online version, at doi:https://doi.org/10.1016/j.jfca.2021.104278. stir bar sorptive extraction method to study different beer styles volatile profiles.
Food Res. Int. 126, 108680.
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