Food Research International 89 (2016) 281–288

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Food Research International

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

Mass spectrometry for the characterization of brewing process

Adriana Fu Vivian, Caroline Tiemi Aoyagui, Diogo Noin de Oliveira, Rodrigo Ramos Catharino ⁎
INNOVARE Biomarkers Laboratory, School of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo 13083-877, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Beer is a carbonated alcoholic beverage produced by fermenting ingredients containing starch, especially malted
Received 1 June 2016 cereals, and other compounds such as water, hops and yeast. The process comprises five main steps: malting,
Received in revised form 10 August 2016 mashing, boiling, fermentation and maturation. There has been growing interest in the subject, since there is in-
Accepted 13 August 2016
creasing demand for beer quality aspects and beer is a ubiquitous alcoholic beverage in the world. This study is
Available online 15 August 2016
based on the manufacturing process of a Brazilian craft brewery, which is characterized by withdrawing samples
Keywords:
during key production stages and using electrospray ionization (ESI) high-resolution mass spectrometry
Beer (HRMS), a selective and reliable technique used in the identification of substances in an expeditious and practical
Brewing way. Multivariate data analysis, namely partial least squares discriminant analysis (PLS-DA) is used to define its
Processomics markers. In both positive and negative modes of PLS-DA score plot, it is possible to notice differences between
Mass spectrometry each stage. VIP score analysis pointed out markers coherent with the process, such as barley components
PLS-DA ((+)-catechin), small peptide varieties, hop content (humulone), yeast metabolic compounds and, in matura-
tion, flavoring compounds (caproic acid, glutaric acid and 2,3-butanediol). Besides that, it was possible to identify
other important substances such as off-flavor precursors and other different trace compounds, according to the
focus given. This is an attractive alternative for the control of food and beverage industry, allowing a quick assess-
ment of process status before it is finished, preventing higher production costs, ensuring quality and helping the
control of desirable features, as flavor, foam stability and drinkability. Covering different classes of compounds,
this approach suggests a novel analytical strategy: “processomics”, aiming at understanding processes in detail,
promoting control and being able to make improvements,
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Beer is a carbonated alcoholic beverage produced by fermenting in-
gredients containing starch, mainly malted grains such as barley and
wheat (Beltramelli, 2013). In addition to cereals, beer has water, hops
and yeasts, and may include other ingredients such as fruits, herbs
and brewing adjuncts (Sleiman & Venturini Filho, 2008). In general,
beers are composed of carbohydrates, minerals, vitamins, polyphenols
and amino acids (Quifer-Rada et al., 2015). The beer production process,
regardless of the batch size and style, involves the steps of malting,
mashing, wort production, filtration, boiling, fermentation, maturation
and bottling. The considerable interest surrounding beer is justifiable:
it is one of the most widespread alcoholic beverages and is the fifth
most consumed beverage in the world, standing only behind tea, coffee,
milk and soda (Fillaudeau, Blanpain-Avet, & Daufin, 2006). Despite its
millenary tradition, popularity and varieties, the quantification and
identification of beer components and ingredients still represent a chal-
lenge due to the high complexity of the beer matrix, lack of commercial
standard, instability of ingredients and structural similarity of the
⁎ Corresponding author.
E-mail address: rrc@fcm.unicamp.br (R.R. Catharino).
constituents (Ivanova & Spiteller, 2014). The main classification used
is according to fermentation process. There are two large groups,
which may be divided into Ales and Lagers. Ale beers are also known
as top fermenting because its fermentation is at the top of the tanks.
The lagers are known as bottom fermenting, since fermentation takes
place at the bottom of the tanks (Beltramelli, 2013).
In the malting process, starts the production of hydrolytic enzymes
during germination, such as enzymes that break down starch (α-amy-
lase and β-amylase), the cell walls (β-glucanases) and proteins (car-
boxypeptidases and endopeptidases). Then, to stop the enzymatic
activity, the grains are dried at above 85 °C, ranging up to more than
200 °C depending on the aimed malt flavor and color (Baxter &
Hughes, 2001). The drying process also has the function of removing
volatiles that may contribute to the emergence of off-flavors in the
final product and help in grain conservation, since it eliminates all the
moisture (Morado, 2009).
The next step involves grinding the malted grains in a mill. The
crushed malt goes under heating to gelatinize the starch and make it
more susceptible to enzymatic attack. The wort is the result of the
breakdown of amylose and amylopectin molecules into small sugars,
as maltose and glucose. The wort is also rich in soluble proteins, pep-
tides and amino acids, important elements in the stability and quality

http://dx.doi.org/10.1016/j.foodres.2016.08.008
0963-9969/© 2016 Elsevier Ltd. All rights reserved.
282 A.F. Vivian et al. / Food Research International 89 (2016) 281–288
of beer (Baxter & Hughes, 2001). There is no exact definition of the in-
tervals and temperature in wort production, which is a characteristic
of each beer and varies according to the desired result. Nevertheless,
four temperature ranges may be set for the process, based on enzymatic
activation range, as follows (Morado, 2009):
• 40–45 °C: Enzymatic activation. Beta-glucanase enzymes become ac-
tive.
• 50–55 °C: Proteolysis.
• 60–72 °C: Action of alpha and beta-amylase.
• 76–78 °C: Enzymatic inactivation.
Then, it is the moment to add hop pellets and bring to boiling tem-
perature, causing the following effects: sterilization of the wort, enzyme
inactivation, water evaporation (concentration of the wort) and coagu-
lation of proteins. This process is also important for extraction of α- and
β- hops acids, responsible for beer bitterness, flavor and antiseptic ac-
tion (Bernotienë, Nivinshiene, Butkienë, & Mochkute, 2004). The next
stage, the fermentation process, is the phase in which sugars are con-
verted into alcohol. This phase depends on many cellular mechanisms,
being influenced by other factors such as temperature, pH, dissolved ox-
ygen, agitation, availability of sugars and other nutrients, the presence
of contaminants and inoculation rate (Bokulich & Bamforth, 2013). Dur-
ing the process, the simplest sugars are fermented first, using glucose,
fructose, sucrose, maltose and maltotriose, in that order (Palmer, 2006).
When the stationary phase of fermentation starts, the majority of
yeasts are removed and then maturation starts. The remaining yeast
promotes two effects: higher carbon dioxide production and chemical
removal of undesirable compounds such as diacetyl, acetaldehyde and
acid sulphide (Yamauchi, Okamoto, Murayama, Kajino, & Noguchi,
1995). Moreover, this phase aims to initiate beer clarification (sedimen-
tation of the yeast cells) and maintain the beer in the reduced state,
preventing oxidation during storage. During maturation, higher alco-
hols and fatty acids do not change significantly. However, other esters
intensify, consisting mainly: ethyl acetate, isoamyl acetate, ethyl
caprylate and ethyl caproate (BENTO, CARVALHO, & Silva, 2007). After
the maturation, the beer must be bottled. Before this process, the re-
maining yeast may be removed and the beer is then cooled and may
be filtered.
Mass spectrometry (MS) is a technique widely used in the identifica-
tion of substances, with several ionization techniques. Great improve-
ments have been made, especially on electrospray ionization (ESI),
which caused the increased sensitivity of equipment and contributed
to the application of MS in a number of different products, contributing
to advances in quality control (QC)(Gillespie & Winger, 2011). The
progress of ESI in recent years is due to technical versatility and specific-
ity, so it can be applied to biomolecules and biomarkers processes found
in complex matrices (Riccio et al., 2010). The simplicity for sample prep-
aration and speed in obtaining fingerprints with several chemical infor-
mation increases its applicability in several areas (Quifer-Rada et al.,
2015). The sensory properties of food and drinks are key characteristics
for customer acceptability and QC. However, in brewing, QC has a sub-
jective evaluation by a combination of visual, flavor and taste percep-
tions that can lead to misunderstandings. Considering this scenario, a
fast, objective, versatile and analytical method such as ESI-MS is desir-
able for quality and process controls (Riccio et al., 2010).
Moreover, current trends in manufacturing processes rely on the
search for variables to predict and clarify performances and events.
When elements are not redundant, have a well-understood connection
and data are not extensive, multiple linear regression is advisable. On
the other hand, the PLS-DA (Partial Least Squares- Discriminant Analy-
sis) algorithm is appropriate when there are multi-collinear data and
much information. It is valuable for categorization purposes and bio-
marker election, presenting the results in a visual and graphical way,
e.g. score plots (Brereton & Lloyd, 2014). In combination with mass
spectrometry fingerprinting, PLS-DA becomes a powerful tool for pro-
cess description and control, identifying the main compounds of any
process. In this trend, the present contribution focuses at demonstrating
the ability of this group of techniques in pointing out markers to follow
up processes based on quality and productivity aspects, as well as
evidencing different compounds of interest such as off-flavors.
2. Materials and methods
2.1. Chemical and reagents
Methanol, ammonium hydroxide and formic acid solutions were
purchased from J. T. Baker (Xalostoc, Mexico) and used with no further
purification. Deionized water was obtained with a Milli-Q system
(Millipore, USA).
2.2. Samples
Samples were obtained from a craft brewery, Cervejaria Nacional,
São Paulo, Brazil. The India Pale Ale (IPA) style was the study focus. Sam-
ples were withdrawn from the end of the following stages: mash, final
wort, boiling, fermentation and maturation. Experiments were carried
out in five replicates. The beer sample has 60 IBUs, 7.5% ABV, and is con-
tains Pilsen, Pale Ale and Cara Munich malt, in addition to Hallertauer
Magnum, Chinook, Cascade and Sincoe hops.
2.3. Sample preparation
The beer samples were stored at 4 °C before analysis for up to 24 h,
protected from light exposure. Samples were prepared by placing
10 μL of beer sample in a 1 mL vial with 990 μL of methanol:water
(1:1). For positive analysis, 10 μL of sample solution were added to
990 μL of methanol:water (1:1) and 1 μL of formic acid. For negative
analysis, the same dilution was adopted, but with the addition of 1 μL
of ammonium hydroxide instead of acid. All samples were filtered be-
fore analysis.
2.4. Equipment
MS fingerprints were obtained using an LTQ-XL Orbitrap Discovery
instrument (Thermo Scientific, California, USA). The injection pump
uses a continuous flow of 10 μL.min−1 and a 500-μL Hamilton Gastight
glass syringe (Hamilton, Nevada, USA). High-resolution mass spectra
were obtained after a period of 30 s, in a scan range from 50 to
2000 m/z, in both positive and negative modes.
2.5. Data management
Fingerprints obtained were processed using MetaboAnalyst 3.0 and
tables of m/z values as functions of intensities were extracted from the
bulk spectra. In order to identify chemical markers of beer production
samples, score plots were obtained after PLS-DA of normalized data in
combination with VIP score analysis considering the five main steps of
beer production (mash (M1), wort (M2), boiling (M3), fermentation
(M4) and maturation (M5)). To evaluate if there were differences con-
sidering the overall process a previous graphical analysis of score plots
was made. For VIP score analysis, data were processed considering the
process sequence, in other words, comparisons were made between
M1–M2, M2–M3, M3–M4 and M4–M5, and signals were considered
markers only if presenting a VIP score greater than 2. No data filtering
and transformation were performed apart from range scaling. Signal
identification was performed using Lipid Maps, FoodDB and Metlin da-
tabases considering a mass error less than 2 ppm.
 A.F. Vivian et al. / Food Research International 89 (2016) 281–288 283

381
100

95
90

85
80
75

70 325

65

60
55 258 360

50

45
116
40
543
35
30
25 130
145
20
156 219 296 522
15 382 474
294 309
10 175 229 326 458
85 544
5
383
M1
0
100 200 300 400 500 600 700 800 900 1000
377 m/z
100

95
90 439
85
80 341
161
75

70
65

60
55
221
50
179
45
40
191
35
379
30 383
25

20 387 503
215 440
15 263 431 470
101 143 342 601
281 339 425 683
10
5
89 128 259 308 474 539
M2
0
100 200 300 400 500 600 700 800 900 1000
377 m/z
100

95
90

85
80
75

70
65

60
55
50

45
40

35
379
30
25

20
15 161
431 539
10
163
215
341 383 503 M3
5
0
100 200 300 400 500 600 700 800 900 1000
191 m/z
100

95
90

85
80 383

75

70
65

60
55
221
50

45
40

35
161
30 339
25
179 259 341 545
20
15
263
195 384 425
10 290 503
5
133
333 377 419 439 M4
0
100 200 300 400 500 600 700 800 900 1000
m/z
223
100
95
90
85 149

80
75
70
65
60
55
50 283

45
40
209
35
30
25
20
153
15 189 357
267 981
343
10 224
297
5 M5
0
100 200 300 400 500 600 700 800 900 1000
m/z

Fig. 1. Representative mass spectra (fingerprints) of each beer production stage (M1 - mash; M2 - wort; M3 - boiling; M4 - fermentation; M5 - maturation.). Negative ion mode. It is
possible to identify anionic species as [M + Cl]− adducts of maltose (m/z 377.06) and matotriose (m/z 379.05); and [M + H]− species for maltose (m/z 341.09), maltotriose (m/z
503.12) and glucose (m/z 179.04).
284 A.F. Vivian et al. / Food Research International 89 (2016) 281–288

381
100
95
90
85
80
75
70
65
60
55
50
45
40 258
35
30
543
25 325
219
133
20 156
15 130 309 360
175 294 382 M1
10 474
205 221 383 544
5
0
100 200 300 400 500 600 700 800 900 1000
381 m/z
100
95
90
85
80
365
75
70
65 175
60
55
50
45
40
35
325
30
25 360
527
20 258 543
15 130 147 382
10 219 309 474 M2
127 203 294 383
5
0
100 200 300 381400 500 600 700 800 900 1000
100 m/z
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20 543
219
15 175
M3
10
383
5
0
100 200 300 400 500 600 700 800 900 1000
175 m/z
100
258
95
90
85
80
325
75
70
65
60
55
50
45
309
40
35 381
30 543
25
294
20 156
145 266
15 337
85 127 182 205
244 471
M4
10
97 234 544 705
5
0
100 200 300 400 500 600 700 800 900 1000
175 m/z
100
95
90
85
80
75
70
65
60
55 258
50
45
40
325
35
30
25 156 309
20 266
381
15 294 543
116 145 182 205 M5
10 268 337
244 527
5
0
100 200 300 400 500 600 700 800 900 1000
m/z

Fig. 2. Fingerprints of each stage of beer production (M1 - mash; M2 - wort; M3 - boiling; M4 - fermentation; M5 - maturation). Positive ion mode. It is possible to identify cationic species
corresponding to [M + K]+ and [M + Na]+ adducts of maltose (m/z 381.10), maltotriose (m/z 543.17) and glucose (m/z 219.04).
 A.F. Vivian et al. / Food Research International 89 (2016) 281–288
3. Results and discussion
Fingerprints obtained are presented in Figs. 1 and 2. PLS-DA results
are presented in Figs. 3 and 4, for negative (neg) and positive (pos)
modes, respectively. In both cases, it is possible to notice statistical dif-
ferences between each stage due to the graphic separation presented
in the score plots. Results after VIP score analysis and components iden-
tification are summarized in Tables 1 and 2 (Jewison et al., 2011; Preedy,
2011; Yannai, 2012) and 2(Collin, Jerkovic, Bröhan, & Callemien, 2013;
Enebo, Blomgren, & Johnsson, 1955; Ge, Usack, Spirito, & Angenent,
2015; Ng, Jung, Lee, & Oh, 2012; Preedy, 2011; Yannai, 2012).
Spectra presented expected signals for beer sugars as cationic spe-
cies, corresponding to [M + K]+ and [M + Na]+ adducts of maltose
(m/z 381.10), maltotriose (m/z 543.17) and glucose (m/z 219.04); an-
ionic species were observed as [M + Cl]− adducts of maltose (m/z
377.06) and matotriose (m/z 379.05); and [M + H]− species for maltose
(m/z 341.09), maltotriose (m/z 503.12) and glucose (m/z
179.04)(Araújo et al., 2005). All of them appear mainly in M2-Wort
and M3-Boiling spectra, with a lower intensity in M4-Fermentation. De-
spite not being a quantitative method, this variation of intensity is co-
herent to what is expected from the process, since fermentation
transforms sugars into alcohol, and thus its concentration tends to
reduce.
In addition to sugars, the statistical analysis using PLS-DA process
pointed out process markers, i.e., compounds that indicate whether
the transformation is occurring based on sample differences. The score
plots comparing all five stages (mash (M1), wort (M2) boiling (M3),
fermentation (M4) and maturation (M5)), show that there is precision
Fig. 3. PLS-DA plots from negative mode spectra illustrate that each stage is different from each other and the replicates are precise between them. M1 - mash; M2 - wort; M3 - boiling; M4 -
fermentation; M5 - maturation.
285
between replicates, since they cluster among each other. As each sample
clusters are far from each other, this illustrates that there are differences
between the steps and, after VIP score analysis, it is possible to find rel-
evant markers.
Different peptides appear as markers of many phases, mainly in M2
(Wort). Considering that in this phase the proteolysis of malt content
happens at 50–55 °C (Morado, 2009), it is coherent with the process, al-
though only generic molecules were observed as markers, as they also
appeared in M3-boiling, M4-Fermentation and M5-Maturation phases.
Despite being generic, amino acids and peptides play important roles
in beer quality and brewing process, especially as foam stabilizing
agents, and flavor and haze development agents (combined with poly-
phenols) in beer. Wort amino acids are the major source of nitrogen for
fermentation yeast, and are consumed according to their proprieties.
Proline, for example, is the last to be consumed, and its presence in
final beer is directly related to product stability (haze) and, due to its re-
action with maltose (through the Maillard reaction mechanism), it may
affect directly the color development in beer. Moreover, there is a direct
correlation among each amino acid, fermentation temperature and the
esters and higher alcohols formed, molecules that are essential to beer
flavor development (Preedy, 2011). The undesirable taste of vicinal
diketones, for instance, is result of valine and isoleucine intermediates
biosynthesis during fermentation process. One method to control
diacetyl production suggested is valine feedback inhibition control
(Krogerus & Gibson, 2013).
Beer phenols also contribute directly to beer flavor, color and haze,
and may be found in hops, barley and malt. During mashing, malt flavo-
noids are dissolved in the wort, but after filtration, they tend to
286 A.F. Vivian et al. / Food Research International 89 (2016) 281–288

Fig. 4. PLS-DA plots from positive mode spectra illustrate that each stage is different from each other and the replicates are precise between them. M1 - mash; M2 - wort; M3 - boiling; M4 -
fermentation; M5 - maturation.

disappear through binding to coagulated proteins, for example. Thus,
(+)-catechin found in M2-Wort is a marker that is originated from
malt grain, and it is the main monomeric unit found, with an important
role since it can induce haze upon storage (Collin et al., 2013). In other
studies, (−)-epicatechin, (−)-catechin gallate and (−)-epicatechin gal-
late and epigallocatechin were also detected (Yannai, 2012). Epigallo-
catechin metabolites were found as markers in M4-Fermentation
phase, and it is suggested to be result of S. cerevisae metabolism and
action of its many O-methyltransferase (Niewmierzycka & Clarke,
1999). Other compounds that are important to beer flavor were found
in hops and appear as markers of M3-Boiling step, such as humulone
and flavone, expressive components of hop pellets (Bernotienë et al.,
2004). Cohulupone, one marker of M5-Maturation, is minor hop con-
stituent and an oxidation product of colupulone. During fermentation
and maturation, it does not undergo any other transformation, possibly
explaining why it appears as a marker in the final beer. It is responsible

Table 1
Markers found after PLS-DA and VIP score analysis- Negative ion mode.

Stage Ion m/z Compound Compound description

(spectra) (δPPM)

[M + 503.1229
Peptide Cys-Leu-Met-Cys
Cl]− (0)
[M + 575.1398 Peptide Asp-Asp-Glu-Tyr Remaining peptides from wort proteolysis. Yeast will consume according to amino acid
Cl]− (2) proprieties (Preedy, 2011).
Boiling (M3)
[M + 472.1725 Peptide Tyr-Glu-Tyr
H]− (2)
[M + 221.0683 Flavone Crystalline compound present in hops (Yannai, 2012)
H]− (1)
Fermentation [M + 545.1339 Peptide Asp-His-Cys-His Remaining peptides from wort proteolysis and not used in yeast metabolism.
(M4) H]− (0)
[M + 443.1064
Peptide Cys-Cys-Gly-Tyr
H]− (0)
Fermentation [M + 425.0946 Epigallocatechin-3-O-(4-hydroxybenzoate) Epigallocatechins are isolated from barley grains, hops and final beer (Yannai, 2012). It is a
(M4) H]− (2) metabolite of S. cerevisae (Jewison et al., 2011), by means of O-methyltransferase enzyme.
[M + 485.1147 Epigallocatechin
H]− (3) 3-O-(3,5-di-O-methylgallate)
 A.F. Vivian et al. / Food Research International 89 (2016) 281–288 287

Table 2
Markers found after PLS-DA and VIP score analysis- Positive ion mode.

Stage Ion m/z Compound Compound description

(spectra) (δPPM)

[M + 366,1330 Peptide
H]+ (1) Met-Thr-Asp
[M + 528,1945 Peptide
H]+ (0) Met-Phe-Gln-Cys
Peptides resulting from proteolysis.
[M + 529,1963 Peptide
H]+ (0) Ser-Glu-Met-Tyr
Wort (M2)
[M + 528,1959 Peptide
H]+ (1) Asn-Arg-Asn-Cys
[M + 291.0795 (+)- Catechin Phenol found in large amount in hops, can be found in small concentration in malt grain (10–100 ppm) and is
H]+ (2) responsible for astringency mouth feel (Collin et al., 2013).
[M + 133.0424 Glutaric Acid Non-volatile organic acid (Enebo et al., 1955).
H]+ (2)
[M + 221,0807
Humulone Main component of hops (Yannai, 2012).
H]+ (2)
[M + 381.1075 Peptide
H]+ (1) Gly-Asp-Cys-Ser
Boiling [M + 382.1101 Peptide
(M3) H]+ (1) Cys-Glu-Met
Remaining peptides from wort. Yeast will consume according to amino acid proprieties (Preedy, 2011).
[M + 366.1330 Peptide
H]+ (1) Met-Glu-Ser
[M + 435.1640 Peptide
K +] (0) Pro-Pro-Pro-Ser
[M + 117,0829
Caproic Acid Result of yeast activity (Ge et al., 2015).
H]+ (1)
[M + 252.1201 2,3- butanediol Result of S. cerevisae metabolism (Ng et al., 2012)
Maturation H]+ (1) glucoside
(M5) [M + 318.1487 Peptide Peptide remaining from raw materials.
H]+ (2) Cys-Val-Pro
[M + 319, Cohulupone Cohulupone is a constituent of hops
H]+ 1825 (2)

* Arg: Arginine; Asn: Asparagine; Asp: Aspartic Acid; Cys: Cysteine; Met: Methionine; Gln: Glutamine; Glu: Glutamic acid; Gly: Glycine; His: Histidine; Phe: Phenylalanine; Ser: Serine;
Thr: Threonine; Tyr: Tyrosine;

for the pleasant bitterness in beer, although it is less potent than
isohumulone (Laws, 1968).
Caproic acid, also direct linked to beer quality and sensorial aspects,
was detected as maker in M5-Maturation. Depending on its concentra-
tion, it may be considered either an off-flavor or a desirable component
in beer (Horák, Čulík, Jurková, Čejka, & Kellner, 2008). Detecting its
quantity during maturation is a way to predict if the final product will
be according to what is expected or not. Another important compound
in maturation that is related to quality is 2,3-butanediol, result of con-
version of diacetyl in acetoin through diacetyl reductase, then reduction
of acetoin by butanediol dehydrogenase (Ng et al., 2012). Diacetyl is an
undesirable off-flavor, and it is important to be converted to 2,3-
butanediol during fermentation and maturation, as the latter has a larg-
er threshold detection. Other markers found were non-volatile organic
acids, compounds present in wort and in larger concentration in the
final beer, due to fermentation metabolism, and some more expressive-
ly in top-fermenting beers (Ales). One example is glutaric acid, which is
the content in final beer that characterizes the fermentation procedure,
specific from Ales (Enebo et al., 1955).
Off-flavor signs are observed during the process. Despite not
being considered a process change marker, they are remarkable to
final quality aspects. The importance of flavor and its components
is not only to give the final quality characteristics to beer, but is
also a strong indicator that the process was successful (Morado,
2009). The anionic species [M + H−] found are p-methane-8-thiol
(M1; m/z 185.1076), a precursor of catty urine flavor and ethyl
mystrate (M3, m/z 255.2396) – an ester with vegetable oil flavor. It
is important to highlight that they appear in early stages (M1 and
M3) which allow to previous evaluate if the process is happening
according to expected, without the necessity of waiting to taste the
final product.
4. Conclusion
Direct injection ESI-HRMS demonstrated to be suitable for a rapid,
simple and robust fingerprinting approach of complex mixtures such
as beer, as well as for pointing out the main chemical players in process.
The technique is convenient for process monitoring, since it requires
small sample size, simple handling and fast injection in a mass spec-
trometer. This is an attractive alternative for the control of food and bev-
erages, particularly when associated with qualitative multivariate
statistics. PLS-DA shows clear differences between the phases in the
overall process and VIP score analysis allowed to identify the main
markers of the process, malt and hop components, in addition to sub-
stances resulting from mashing, fermentation and maturation process-
es, such as humulone, glutaric acid, 2,3-butanediol and caproic acid.
Besides markers, MS is capable of evidencing other important sub-
stances such as off-flavors precursors. Our approach is able to evidence
whether the process is happening according to expected before it is fin-
ished, without subjective sensorial analysis, thereby suggesting a new
area for mass spectrometry application, in a zoom-in ‘processomics’ ap-
proach to better understand any process, and thus control and improve
it. The main contributions for developing further applications is the pos-
sibility of detection of off-flavors before obtaining the final product, pre-
vention of higher production costs and assuring final quality,
monitoring contamination and controlling desirable characteristics,
such as presence of esters and bitter compounds, foam stability and, ul-
timately, drinkability.
Author contributions
A.F.V.: designed and conducted the mass spectrometry experiments,
analyzed the results, wrote the paper and prepared the figures. C.T.A.:
288 A.F. Vivian et al. / Food Research International 89 (2016) 281–288
conducted the mass spectrometry experiments and analyzed the re-
sults. D.N.O. and R.R.C.: idealized all experiments, analyzed the results
and revised the paper.
Acknowledgements
Cervejaria Nacional supported this study. All authors have read and
approved the final manuscript. We also thank FAPESP for the financial
support that allowed this work to be performed (Grants # 2011/
50400-4, 2014/00084-2 and 2015/06809-1).
References
Araújo, A. S., Da Rocha, L. L., Tomazela, D. M., Sawaya, A. C., Almeida, R. R., Catharino, R. R.,
& Eberlin, M. N. (2005). Electrospray ionization mass spectrometry fingerprinting of
beer. Analyst, 130(6), 884–889.
Baxter, E. D., & Hughes, P. S. (2001). Beer: Quality, safety and nutritional aspects. Royal So-
ciety of Chemistry.
Beltramelli, M. (2013). Cervejas: Brejas & Birras. Cidade São Paulo: Editora Leya, Editora
Tainã Bispo.
Bento, C., Carvalho, G., & Silva, J. (2007). Elementos biotecnológicos fundamentais no
processo cervejeiro: 3° parte–A maturação. São Paulo: Revista Analytica, 27.
Bernotienë, G., Nivinshiene, O., Butkienë, R., & Mochkute, D. (2004). Chemical composi-
tion of essential oils of hops (Humulus lupulus L.) growing wild in Aukstaitija.
Chemija, 15(2), 31–36.
Bokulich, N. A., & Bamforth, C. W. (2013). The microbiology of malting and brewing.
Microbiology and Molecular Biology Reviews, 77(2), 157–172.
Brereton, R. G., & Lloyd, G. R. (2014). Partial least squares discriminant analysis: Taking
the magic away. Journal of Chemometrics, 28(4), 213–225.
Collin, S., Jerkovic, V., Bröhan, M., & Callemien, D. (2013). Polyphenols and beer quality.
Natural products (pp. 2333–2359). Springer.
Enebo, L., Blomgren, G., & Johnsson, E. (1955). Low molecular non-volatile organic acids in
wort and beer. Journal of the Institute of Brewing, 61(5), 408–411.
Fillaudeau, L., Blanpain-Avet, P., & Daufin, G. (2006). Water, wastewater and waste man-
agement in brewing industries. Journal of Cleaner Production, 14(5), 463–471.
Ge, S., Usack, J. G., Spirito, C. M., & Angenent, L. T. (2015). Long-term n-caproic acid pro-
duction from yeast-fermentation beer in an anaerobic bioreactor with continuous
product extraction. Environmental Science & Technology, 49(13), 8012–8021.
Gillespie, T. A., & Winger, B. E. (2011). Mass spectrometry for small molecule pharmaceu-
tical product development: A review. Mass Spectrometry Reviews, 30(3), 479–490.
Horák, T., Čulík, J., Jurková, M., Čejka, P., & Kellner, V. (2008). Determination of free medi-
um-chain fatty acids in beer by stir bar sorptive extraction. Journal of Chromatography
A, 1196, 96–99.
Ivanova, B., & Spiteller, M. (2014). Solid-state determination of hop bitter acids in beer by
UV–MALDI–Orbitrap mass spectrometry. Journal of Food Measurement and
Characterization, 8(4), 343–355.
Jewison, T., Knox, C., Neveu, V., Djoumbou, Y., Guo, A. C., Lee, J., ... Sinelnikov, I. (2011).
YMDB: The yeast metabolome database. Nucleic Acids Research (gkr916).
Krogerus, K., & Gibson, B. R. (2013). Influence of valine and other amino acids on total
diacetyl and 2, 3-pentanedione levels during fermentation of brewer's wort. Applied
Microbiology and Biotechnology, 97(15), 6919–6930.
Laws, D. (1968). Hop resins and beer flavour V. The significance of oxidized hop resins in
brewing. Journal of the Institute of Brewing, 74(2), 178–182.
Morado, R. (2009). Larousse de cerveja: Larousse do Brasil.
Ng, C. Y., Jung, M. -y., Lee, J., & Oh, M. -K. (2012). Production of 2, 3-butanediol in Saccha-
romyces cerevisiae by in silico aided metabolic engineering. Microbial Cell Factories,
11(1), 1.
Niewmierzycka, A., & Clarke, S. (1999). S-Adenosylmethionine-dependent methylation in
Saccharomyces cerevisiae identification of a novel protein arginine methyltransferase.
Journal of Biological Chemistry, 274(2), 814–824.
Palmer, J. J. (2006). How to brew. EUA: Brewers Publications.
Preedy, V. R. (2011). Beer in health and disease prevention. Academic Press.
Quifer-Rada, P., Vallverdú-Queralt, A., Martínez-Huélamo, M., Chiva-Blanch, G., Jáuregui,
O., Estruch, R., & Lamuela-Raventós, R. (2015). A comprehensive characterisation of
beer polyphenols by high resolution mass spectrometry (LC–ESI-LTQ-Orbitrap-MS).
Food Chemistry, 169, 336–343.
Riccio, M. F., Saraiva, S. A., Marques, L. A., Alberici, R., Haddad, R., Moller, J. C., ... Catharino,
R. R. (2010). Easy mass spectrometry for metabolomics and quality control of vegeta-
ble and animal fats. European Journal of Lipid Science and Technology, 112(4), 434–438.
Sleiman, M., & Venturini Filho, W. (2008). Relação entre percentual de malte e preço em
cervejas tipo pilsen. Revista Energia na Agricultura, 23(1), 98–108.
Yamauchi, Y., Okamoto, T., Murayama, H., Kajino, K., & Noguchi, K. (1995). Rapid matura-
tion of beer using an immobilized yeast bioreactor. 2. Balance of total diacetyl reduc-
tion and regeneration. Journal of Biotechnology, 38(2), 109–116.
Yannai, S. (2012). Dictionary of food compounds with CD-ROM. CRC Press.