You are on page 1of 6

Food Chemistry 215 (2017) 341–346

Contents lists available at ScienceDirect

Food Chemistry
journal homepage:

Analytical Methods

Determination of free fatty acids in beer

Elisabetta Bravi a,⇑, Ombretta Marconi a, Valeria Sileoni b, Giuseppe Perretti b
Italian Brewing Research Center (Centro di Eccellenza per la Ricerca sulla Birra – CERB), University of Perugia, Via S. Costanzo n.c.n., 06126 Perugia, Italy
Department of Agricultural, Food and Environmental Sciences, University of Perugia, Via S. Costanzo, n.c.n., 06126 Perugia, Italy

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

Article history: Free fatty acids (FFA) content of beer affects the ability to form a stable head of foam and plays an
Received 29 October 2015 important role in beer staling. Moreover, the presence of saturated FAs is related sometimes to gushing
Received in revised form 13 July 2016 problems in beer. The aim of this research was to validate an analytical method for the determination of
Accepted 28 July 2016
FFAs in beer. The extraction of FFAs in beer was achieved via Liquid–Liquid Cartridge Extraction (LLCE),
Available online 29 July 2016
the FFAs extract was purified by Solid Phase Extraction (SPE), methylated by boron trifluoride in
methanol, and injected into GC-FID system. The performance criteria demonstrate that this method is
Chemical compounds studied in this article:
suitable for the analysis of medium and long chain FFAs in beer. The proposed method was tested on four
Caprylic acid (PubChem CID: 379)
Capric acid (PubChem CID: 2969)
experimental beers.
Lauric acid (PubChem CID: 3893) Ó 2016 Elsevier Ltd. All rights reserved.
Myristic acid (PubChem CID: 11005)
Myristoleic acid (PubChem CID: 5281119)
Palmitic acid (PubChem CID: 985)
Palmitoleic acid (PubChem CID: 445638)
Stearic acid (PubChem CID: 5281)
Oleic acid (PubChem CID: 445639)
Linoleic acid (PubChem CID: 5280450)
Linolenic acid (PubChem CID: 5280934)

Free fatty acids

1. Introduction length increased and that the unsaturated FAs are more hydrophilic
compared with the saturated with the same carbon-chain length
Although free fatty acids (FFA) are quantitatively only minor con- (double bonds are more hydrophilic than single bonds). Therefore,
stituents of beer their presence has long been considered to have an unsaturated FAs absorbs more weakly at the surface of gas-
adverse effects on beer quality. A stable foam is an important aspect bubbles than a saturated FAs of the same chain length (Dickie,
of beer quality as perceived by the consumer (Segawa, Yamashita, Cann, Norman, Bamforth, & Muller, 2001; Segawa et al., 2002).
Mitani, & Masachika, 2002). Beer contains foam-positive com- Beer consists of many compounds (volatile and non-volatile) that
pounds (proteins and iso-a-acids) and foam-negative compounds affect beer flavor, many of these aroma compounds are synthesized
(alcohols and FFAs). FFAs weaken the foam film by absorbing on by yeasts during fermentation, others derive directly from the raw
the gas bubble surface or by interaction with foam-generating pro- materials. FFAs represent one group of these compounds that affect
teins. Moreover, the detrimental effect caused by the different FFAs beer taste, and also the foam stability of beer (Bamforth, 1985;
is related to their surface absorption properties. Considering that the Clapperton, 1978; Horak et al., 2009; Meilgaard, Dalgliesh, &
molecular hydrophobicity of the saturated FAs increased as chain- Clapperton, 1979). Medium-chain fatty acids such as hexanoic,
octanoic, and decanoic acids, responsible for rancid or goaty flavor
characteristics, are formed by yeasts during fermentation, and their
⇑ Corresponding author. formation is influenced by yeast strain, original gravity, wort com-
E-mail address: (E. Bravi). position, and degree of aeration (Horak et al., 2009). Off-flavors
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
342 E. Bravi et al. / Food Chemistry 215 (2017) 341–346

due to these acids normally arise from excess formation during fer- FAs, the SBSE is a time-consuming procedure, the SPE technique
mentation and not for other causes such as infection or raw materi- is quite fast and lead good recoveries and reproducibility. Never-
als. Since the aroma contributions for medium-chain FFAs are theless, no validation procedure was carried out and the suitability
additive, the rancid and goaty off-flavors can occur in beer even of the method for the quality control for the brewing process and
though none of the individual FAs are present above threshold con- the assessment of the final product quality not guaranteed.
centrations (Horak et al., 2009). Long-chain unsaturated FAs, such as In order to determine low amounts (ppm) of FFAs in a aqueous
linoleic and linolenic acids, directly deriving from raw materials or matrix, such as beer samples, the development of a simple,
from the yeast metabolism, are of great importance because their accurate and efficient extraction method which avoids emulsion
oxidative degradation may lead to the formation of a characteristic formation and which allows the simultaneous extraction of a wide
aging flavor (Bravi, Perretti, Buzzini, Della Sera, & Fantozzi, 2009; range of fatty acids (with different chain length) is required. The
Horak et al., 2009; Kaneda, Kano, Kamimura, Osawa, & Kawakishi, same authors reported on the development of a method for the
1990; Vanderhaegen, Nevin, Verachtert, & Derdelinckx, 2006) and, determination of medium and long chain FFAs in beer wort using
although their level is normally very low in beer, an unexpected a sorbent assisted liquid–liquid extraction (by the use of the inert
increase, coupled with wrong storage conditions, can result in unde- support of ChemElut cartridges) that, avoiding emulsion formation,
sirable stale flavor (Vanderhaegen et al., 2006). Finally, saturated facilitate efficient extraction of small amount of FFAs. In order to
FAs, derived from both raw materials and yeast, seem to be related evaluate the quality and to predict the stability of beer, by modify-
to the spontaneous over foaming (gushing) in beer (Carrington, ing the method developed for the beer wort, the authors developed
Collett, Dunkin, & Halek, 1972; Christian, Titze, & Ilberg, 2011; and validate a reliable method for the quantification of FFAs in
Muller, Schmid, Becker, & Gastl, 2010). beer. The proposed method was tested on four experimental beers.
Due to their involvement in different aspects of beer quality,
simple and reliable methods for analyzing FFAs content and profile
in beer are desirable. Both HPLC and GC method has been used for 2. Experimental methods
the determination of FAs in beer (Bravi, Perretti, Montanari, Favati,
& Fantozzi, 2007; Irwin & Thompson, 1987; Kaneda et al., 1990; 2.1. Materials
Kobayashi, Kaneda, Kano, & Koshino, 1993; Ng, 2002; Van der
Meersche, Devreux, & Masschelein, 1979). Chromatographic meth- The unmalted and the flocked T. monococcum (emmer), variety
ods for analyzing fatty acids required a sample preparation step. Monlis, both dehulled, samples were supplied by Prometeo srl
The extraction is a very critical step which often needs complex (Urbino, Italy).
procedures resulting in low recoveries. In the past, the approaches Twenty commercial beer samples of the same brand and batch
to the problem of extraction and concentration of the FFAs in beer were purchased from local market and were used for the develop-
have been based on steam distillation (Irwin & Thompson, 1987; ment and validation of the method. Beer experimental samples
Van der Meersche et al., 1979) and liquid-liquid solvent extraction were supplied by CERB (Italian Brewing Research Centre, University
techniques (Chen, Jamieson, & Van Gheluwe, 1980; DeVries, 1990; of Perugia) and were used to test the developed method. Amber
Kaneda et al., 1990; Kobayashi et al., 1993; MacPherson & Buckee, beer sample (AB) was obtained using a mixture of different malts,
1974; Ng, 2002; Taylor & Kirsop, 1977). The steam distillation tech- 80% of Pilsner, 15% of CararedÒ, and 5% of CaramunichÒ (Weyer-
niques are time-consuming and labor intensive. Liquid-liquid mann, Germany); blond beer sample (BB) was obtained using 80%
extraction procedures suffer from problems of emulsion formation, of Pilsner, 10% of CarapilsÒ, 5% of CarahellÒ (Weyermann, Ger-
responsible for analyte loss; moreover, they are complex, time- many), and 5% of crude wheat (Prometeo srl, Italy); an emmer beer
consuming and labor-intensive and require large volumes of sample (EB) was obtained using 75% of Pilsner, 15% of CarapilsÒ
organic solvents. On the other hand, the extraction of samples with (Weyermann, Germany), and 10% of crude emmer (Prometeo srl,
small volumes of solvent results in poor reproducibility and Italy); finally, an emmer malt beer (EMB) was obtained using a mix-
extractability of some FAs because of their low solubility in the sol- ture of two malts obtained by experimentally malted emmer, 95%
vent (Bravi, Benedetti, Marconi, & Perretti, 2014; Hawthorne, Jones, of Pils emmer and 5% of Caramel emmer. Emmer was malted in
Barrett, Kavanagh, & Clarke, 1986; Horak et al., 2009). an automatic micromalting system from Custom Laboratory
Some authors developed more modern procedures, based on Products (Scotland) following the procedure described by Mayer,
the use of solid-phase extraction (SPE). These procedures are quite Marconi, Perretti, Sensidoni, and Fantozzi (2011).
easy and fast, if compared with the previously mentioned method- Two liters of each experimental beer sample were use for the
ologies, reproducible and allow to minimizing solvent consuming. application of the developed method, all the experimental beer
Battistutta, Buiatti, Zenarola, and Zironi (1994) reported on the samples were unfiltered.
developed of a method for the determination of medium chain The experimental beer samples were prepared as follows:
FAs in beer by SPE, thus excluding the long chain FAs which are mashing was performed in a 110 L pilot plant and was carried
also potentially dangerous for beer flavor. Schutz and Back out on by infusion for all the considered beer samples.
(2005) developed a method for the analysis of long chain FAs by For AB the temperature profile of mashing was as follows: (i)
SPE, thus excluding the medium chain FAs (potentially dangerous first steady-state at 52 °C for 5 min, (ii) first rise of temperature
for beer flavor and foam). Other authors (Horak, Culik, Jurkova, from 52 to 65 °C in 12 min, (iii) second steady-state at 65 °C for
Cejka, & Kellner, 2008) reported on the Stir Bar Sorptive Extraction 46 min, second rise of temperature from 65 to 72 °C in 8 min, (iv)
(SBSE) of medium chain FAs in beer, thus excluding the long chain third steady-state at 72 °C for 20 min, (v) final rise from 72 to
ones. Moreover, low recoveries of short chain FAs and an interfer- 76 °C in 5 min, and final steady state at 76 °C for 5 min.
ence by ethanol concentration of beer, that could reduce the versa- For BB the temperature profile of mashing was: (i) first steady-
tility of the method, were observed. Horak et al. (2009) discussed state at 52 °C for 10 min, (ii) first rise of temperature from 52 to
the advantages and the limitations of three different extraction 65 °C in 13 min, (iii) second steady-state at 65 °C for 44 min, sec-
methods (SPE, used as reference, SPME, and SBSE) for the determi- ond rise of temperature from 65 to 72 °C in 7 min, (iv) third
nation of FFAs in beer. For all the three methods, the medium chain steady-state at 72 °C for 20 min, (v) final rise from 72 to 78 °C in
were determined as free fatty acids while the long chain as methyl 10 min, and final steady state at 78 °C for 3 min.
esters, so two run into GC-FID system were needed. The SPME is For EMB the temperature profile of mashing was as follows: (i)
limited because allows only the determination of medium chain first steady-state at 52 °C for 30 min, (ii) first rise of temperature
E. Bravi et al. / Food Chemistry 215 (2017) 341–346 343

from 52 to 67 °C in 10 min, (iii) second steady-state at 67 °C for for 3 min. The carrier gas (H2) flow rate was 1.7 ml/min. Constant
15 min, second rise of temperature from 67 to 72 °C in 10 min, pressure mode was set up. The split ratio was set at 50:1. The tem-
(iv) third steady-state at 72 °C for 20 min, (v) final rise from 72 peratures of injector and of detector were 270 °C and 280 °C,
to 76 °C in 5 min, and final steady state at 76 °C for 10 min. respectively. Peak areas were measured by using an Agilent MSD
For EB the temperature profile of mashing was: (i) first steady- Chemstation for HRGC-FID.
state at 52 °C for 10 min, (ii) first rise of temperature from 52 to
65 °C in 14 min, (iii) second steady-state at 65 °C for 45 min, sec- 2.4. Extraction procedure
ond rise of temperature from 65 to 72 °C in 6 min, (iv) third
steady-state at 72 °C for 20 min, (v) final rise from 72 to 76 °C in Beer samples were degassed and concentrated in a rotary evap-
8 min, and final steady state at 76 °C for 10 min. orator (LABOROTA SEM-320, Resona Technics, Gossau, Switzer-
For all the considered beer samples, after the step of mashing, land) before the FAs determination.
the mash was filtered in a lauter tun. Sparging was executed twice
with 3 L and once with 2 L of brewing water at 78 °C. Next, the 2.4.1. Liquid–liquid cartridge extraction procedure
mash was boiled for 60 min until reaching approximately 12°P. The extraction was carried using porous diatomaceous earth
At the beginning of this phase, hop pellets (Columbus, Centellian (ChemElut cartridges). A sample of 40 ml of concentrated beer
and Summit variety, for RB; Saphir and Cascade variety, for BB; was loaded on a dry ChemElut cartridge and, after 15 min, the col-
Saphir variety, for EMB; and Saaz variety for EB), were added. After umn was washed with two 20 ml portions and with two 10 ml por-
boiling and elimination of the hot trub, the wort was cooled to tion of n-hexane/diethyl ether (1/1; v/v) to extract the lipid
15 °C. Aeration of the wort was achieved. fraction. The effluents, containing the lipid fractions, were col-
For AB the pitching was carried out at 20 °C, by using a commer- lected in a 100 ml bottom flask and evaporated to dryness under
cial yeast (Safale Fermentis, S-05, Marcq-en-Baroeul Cedex, vacuum at 37 °C. The purification and the esterification of the FFAs
France), 70 g/hL (dry weight) after rehydration with sterile water. portion were conducted following the procedure reported by Bravi
The fermentation temperature was maintained at 20 °C for 3 days. et al. (2014) for the determination of FFAs content in beer wort.
For BB the pitching was carried out at 20 °C, by using a commer- The extracted lipids, dissolved in chloroform, were loaded in a
cial yeast (Safale, Fermentis, US-05, Marcq-en-Baroeul Cedex, aminopropyl cartridge previously conditioned with 4 ml of n-
France), 70 g/hL (dry weight) after rehydration with sterile water. hexane. Then, 4 ml of chloroform-isopropanol (2/1; v/v) were
The fermentation temperature was maintained at 20 °C for 7 days. added to remove all the neutral lipids, and the FFA fraction was
For EB and EMB samples the pitching was carried out at 20 °C, eluted with 4 ml of 2% acetic acid in diethyl ether. The methylation
by using a commercial yeast (Safale, Fermentis, WB-06, Marcq- was performed using 1 ml of BF3 methanolic solution (13–15%) at
en-Baroeul Cedex, France), 70 g/hL (dry weight) after rehydration 100 °C for one minute. The FAME fraction was dissolved in 1 ml of
with sterile water. The fermentation temperature was maintained IS solution and injected into GC-FID system.
at 20 °C for 3 days.
For all the beer samples, at the end of fermentation, after cool-
2.5. Validation
ing and when the final attenuation was achieved, the maturation
lasted for a week at 4 °C. At the same time, the discharge of the
Three related performance characteristics, trueness (how close
yeast from the bottom of the tank was performed. After this phase
the mean of ten replication is to a reference value), precision
of maturation, the beer was refermented in the bottle with the
(how close results are to one another), and uncertainty (is an inter-
addition of sucrose (4.5 g/L) and placed in a thermostat at 22 °C
val associated with a measurement result which expresses the
for 2 weeks. At the end of the re-fermentation, the beer was stored
range of values that can reasonably be attributed to the quantity
at 4–5 °C until analysis.
being measured) were used to describe the quality of the results
obtained with the developed method (AOAC International, 2013;
2.2. Other materials
Eurachem Group, 2014). Each step of the entire methodology (con-
centration of beer sample, liquid–liquid cartridge extraction, SPE
HPLC grade n-hexane, diethyl ether, methanol, chloroform,
purification, methylation, and GC-FID analysis) was replicated ten
2-propanol, RPE grade glacial acetic acid, boron trifluoride (BF3)
times in order to validate the method.
methanol complex solution (13–15%), GC standards, SupelcoÒ 37
component FAME MIX, caprylic acid (P99%), margaric acid
2.6. Statistical analysis
(P98%), linoleic acid (P99%), oleic acid (P99%), and methyl non-
adecanoate (P98%) were purchased from Sigma-Aldrich S.r.l.
The FFAs determination on different experimental beer samples
(Milan, Italy). Internal Standard (IS) solution was prepared by
was performed in triplicate and the data were analyzed using
diluting accurately weighed amounts (approx. 10 mg) of methyl
MATLAB Statistics Toolbox (version 7.6.0, The Mathworks Inc., Nat-
nonadecanoate to 200 ml with hexane. Diatomaceous earth car-
ick, MA, USA) to perform the appropriate statistical tests (One-Way
tridges (ChemElut), SPE C18 cartridges (SampliQ C18), and SPE
and Two-Ways ANOVA). Values were considered significantly dif-
amino cartridges (SampliQ aminopropyl) were obtained from
ferent at P < 0.001.
Agilent Technologies (Milan, Italy). A VAC Elut 20 Tall Glass
Manifold (Agilent Technologies, Milan, Italy) was used.
3. Results and discussion
2.3. Equipment and chromatographic conditions
The method development, its validation for the determination
An Agilent Model 6850 gas chromatograph equipped with a of FFAs in beer and its application as study case are reported.
Flame Ionization Detector (FID), a capillary inlet system, a DB-23
(60 m  0.25 mm  0.25 lm) column, and a Model Maestro MPS 3.1. Method development and validation
2XL multipurpose sampler with a 10 ll syringe (Gerstel Inc., Balti-
more, MD) was employed. The programmed oven temperature Since the extraction is the critical time-consuming step in the
was: 130 °C for 1 min, 130–170 °C at 6.5 °C/min, 170–215 °C at available methods (Chen et al., 1980; DeVries, 1990; Hawthorne
2.75 °C/min, 215 °C for 12 min, 215–230 °C at 40 °C/min, 230 °C et al., 1986; Horak et al., 2009; Irwin & Thompson, 1987; Kaneda
344 E. Bravi et al. / Food Chemistry 215 (2017) 341–346

et al., 1990; Kobayashi et al., 1993; MacPherson & Buckee, 1974; Guide, 2014). The obtained results are reported in Table 2. The
Ng, 2002; Taylor & Kirsop, 1977; Van der Meersche et al., 1979), trueness values ranged between 77% and 104%. The recovery
in the proposed method the FFAs were extracted from the beer acceptability is function of the analyte concentration and of the
samples using a single step extraction procedure with ChemElut purpose of the analysis. In the present study (considering the
cartridges. The several advantages of these columns are previously ppm content of FFAs in beer) the recovery limit it is expected to
described by the same authors for the beer wort analyses (Bravi be between 80 and 115%, according to the AOAC (2002). Therefore,
et al., 2014). In order to improve the extraction of medium chain the results we obtained are in the requested range, only two data
fatty acids, present in beer in higher amount then in wort and points for caprylic acid were lower than 80%, but the mean of the
responsible for rancid or goaty flavor characteristics (Horak et al., recoveries for this fatty acid was 80%, which fall in the range of
2009), a mixture of n-hexane/diethyl ether was used with ChemE- acceptability.
lut cartridges. The saturated medium chain FAs (as octanoic, The within-laboratory precision was tested by analyzing beer
decanoic, and dodecanoic acids) have a small polar portion and a samples fortified with 0.4, 1, and 2 mg/L of the four different med-
larger non-polar rod-like hydrocarbon chain that makes these ium and long chain fatty acids ten times. The relative standard
molecules behave more like a non-polar compound. Thus, the deviation of repeatability (%RSDr) values were calculated from
non-polar eluent n-hexane promote the elution of saturated short these data. The results are shown in Table 2. The precision ranged
and medium chain FAs. Different ratio of n-hexane/diethyl ether from 0.4% to 2.0% and is within acceptability criteria for %RSD (±2%)
were tested to verify the efficiency of the solvent mixture and of (Ahuja & Scypinski, 2001; Food and Drug Administration, ORA
the extraction method; the commercial beer sample was fortified Laboratory Procedure, 2014; Shabir, 2003; Sistla, Tata, Kashyap,
with 2 mg/L of caprylic, margaric, oleic and linoleic acid (saturated Chandrasekar, & Diwan, 2005).
medium and long chain, unsaturated and polyunsaturated long The uncertainty of the developed method was determined cal-
chain FAs) and the recoveries were determined by analyzing each culating the Horwitz ratio value (HorRat), the relative results are
of spiked samples six times. The mean percentage recoveries of reported in Table 2. The HorRat is a variation to the Horwitz equa-
each spiking fatty acid extracted with the different extraction mix- tion to be used on single-laboratory validation study (AOAC
ture tested are reported in Table 1. It is evident that the best sol- International, 2013). The predicted repeatability relative standard
vent mixture for the extraction of FFAs of beer is n-hexane/ deviation (PRSDr) for the levels analyzed was calculated according
diethyl ether, 50/50 (v/v), that allows the higher recovery percent- to the Horwitz equation: PRSDr = 2C0.1505, where C is the mass
ages of all tested FAs (78% caprylic, 77% heptadecanoic, 104% oleic, fraction. The ratio of the RSDr calculated from the data to the
and 102% linoleic acid, respectively). The use of ChemElut car- PRSDr calculated from the Horwitz equation results in the HorRat
tridges allowed therefore the rapid and solvent-saving extraction value: HorRat = RSDr/PRSDr. Accepted values are between 0.3 and
of lipids from the aqueous beer matrix, after the extraction the 1.3, under repeatability conditions. The acceptability of the HorRat
FFAs were separated from the lipid extract by SPE, methylated values for within-laboratory relative standard deviations on the
and injected to GC-FID system. low side (values <0.3) may indicate unreported averaging or excel-
The trueness of the method was determined by replicate analy- lent training and experience (AOAC International, 2013).
sis (n = 10) of test sample unspiked and spiked with the four differ- The system suitability, expressed as the %RSD of retention time
ent FAs (caprylic, margaric, oleic and linoleic acid) at three and peak area for each FAME, the range of linearity, the LOD, deter-
different concentrations (0.4, 1, and 2 mg/L of beer). The trueness mined as three times the signal-to-noise ratio, and the LOQ, as ten
was calculated to compare the difference between mean spiked times the signal-to-noise ratio, of each FAME were calculated and
value and mean value with the added concentration of analyte reported in the analysis of FFAs in wort by GC-FID by the same
and was expressed as the relative spike recovery, R0 % (Eurachem authors (Bravi et al., 2014).

Table 1
Recovery of fatty acids, based on one concentration level (2 mg/L) fatty acids spiked in beer, as a function of different solvent mixture.

Fatty acids Extraction mixture

Diethyl ether n-hexane/diethyl ether (30/70, v/v) n-hexane/diethyl ether (50/50, v/v) n-hexane/diethyl ether (70/30, v/v)
Recovery % (mean ± SD) Recovery % (mean ± SD) Recovery % (mean ± SD) Recovery % (mean ± SD)
Caprylic acid (C8:0) 25.2 ± 0.3 35.0 ± 0.0 77.5 ± 0.0 50.0 ± 0.1
Margaric acid (C17:0) 55.6 ± 0.1 57.3 ± 0.3 77.3 ± 0.2 61.7 ± 0.1
Oleic acid (C18:1) 63.0 ± 0.0 78.8 ± 0.2 104.0 ± 0.0 69.9 ± 0.1
Linoleic acid (C18:2) 76.0 ± 0.1 79.6 ± 0.4 101.9 ± 0.1 75.2 ± 0.1

Mean of six replications; SD, standard deviation.

Table 2
Trueness and precision and Horwitz ratio (HorRat) of the optimized method, based on three concentration levels (0.4, 1 and 2 mg/L) of fatty acids spiked in beer.

Fatty acids Concentration level (mg/L)

0.4 1 2
Trueness R0 % Precision % HorRat Trueness R0 % Precision % HorRat Trueness R0 % Precision % HorRat
(mean ± SD) RSDr (mean ± SD) RSDr (mean ± SD) RSDr
Caprylic acid (C8:0) 77 ± 1 1.9 0.1 86 ± 2 2.0 0.1 78 ± 1 1.2 0.1
Margaric acid (C17:0) 80 ± 2 2.0 0.1 81 ± 1 1.1 0.1 81 ± 1 0.9 0.1
Oleic acid (C18:1) 83 ± 1 1.0 0.1 94 ± 4 1.2 0.1 104 ± 3 1.4 0.1
Linoleic acid (C18:2) 84 ± 2 0.4 0.1 96 ± 2 0.7 0.1 102 ± 0 1.2 0.1

Mean of ten replications. SD, standard deviation; R0 , recovery; RSD, relative standard deviation; HorRat, Horwitz ratio.
E. Bravi et al. / Food Chemistry 215 (2017) 341–346 345

Table 3
Free fatty acids patterns of the different experimental beer samples.

Fatty acids AB BB EMB EB

Mean ± SD (mg/L) Mean ± SD (mg/L) Mean ± SD (mg/L) Mean ± SD (mg/L)
Caprylic acid (C8:0) nd 0.0363a ± 0.0011 0.0368a ± 0.0014 0.0483b ± 0.0019
Capric acid (C10:0) 0.0442a ± 0.0008 0.0872b ± 0.0006 0.3086c ± 0.0013 0.0937d ± 0.0022
Lauric acid (C12:0) 0.1192a ± 0.0008 0.4951c ± 0.0011 0.3570b ± 0.0014 0.5593d ± 0.0015
Myristic acid (C14:0) 0.1163a ± 0.0007 0.1195a ± 0.0008 0.1380b ± 0.0012 0.1715c ± 0.0030
Myristoleic acid (C14:1) 0.0431a ± 0.0021 0.0714b ± 0.0009 0.0712b ± 0.0020 0.0733b ± 0.0017
Palmitic acid (C16:0) 0.9814b ± 0.0006 0.9296a ± 0.0022 1.8188d ± 0.0257 1.3165c ± 0.0015
Palmitoleic acid (C16:1) 0.0513a ± 0.0010 0.0731b ± 0.0021 0.0951c ± 0.0028 0.0792b ± 0.0017
Stearic acid (C18:0) 0.8820a ± 0.0019 1.1875b ± 0.0057 1.6850c ± 0.0034 1.6664c ± 0.0220
Oleic acid (C18:1) 0.9353a ± 0.0012 1.3246b ± 0.0200 2.3723c ± 0.0051 2.4378d ± 0.0035
Linoleic acid (C18:2) 2.4649b ± 0.0016 3.7143d ± 0.0079 1.8807a ± 0.0036 3.3900c ± 0.0021
Linolenic acid (C18:3 x3) 0.8142a ± 0.0011 1.3430b ± 0.0157 nd 1.8006c ± 0.0271

Mean of three replications. SD, standard deviation; values in the same row followed by different superscript letters are statistically different (P < 0.001). AB, amber beer; BB,
beer with 5% of crude wheat; EMB, emmer malt beer; EB, beer with 10% of crude emmer; nd, not detectable.

Considering the performance criteria, the developed method et al., 2009), probably influenced the oleic content of final beer,
can offer an useful tool for quality control in brewing industries. as well as, the yeast strain and its metabolism that also deeply
Considering the influence of the raw materials on the FA pattern affect the FAs content in beer (Bravi, Perretti, et al., 2009). For
of finished beer (Bravi, Sensidoni, Floridi, & Perretti, 2009), in the the crude emmer added beer (EB), linoleic acid (3.390 mg/L) was
study case the proposed method was tested on experimental beers the major FA, followed by oleic (2.438 mg/L), linolenic (1.800 mg/
brewed with different raw materials (barley malt, malted emmer, L), stearic (1.666 mg/L), palmitic (1.317 mg/L), and lauric
crude wheat, and crude emmer). (0.559 mg/L) acids. The presence of crude emmer may influence
the FAs level in final beer, in fact the linoleic acid is the major con-
3.2. Application of developed method stituent of the lipids of T. dicoccum, followed by oleic acid. The oleic
acid, in particular, was found at higher percentage (26%) in emmer
The method developed in this study was employed for the than in barley malt (12%) (Anness, 1984; Bravi, Marconi, Perretti, &
determination of FFAs in four different experimental beer samples Fantozzi, 2012; Suchowilska et al., 2009).
(AB, BB, EMB, and EB). The results are reported in Table 3. The FFA
content and profile of beer is affected by the metabolically active
4. Conclusions
yeast cells dispersed in unfiltered beers, as well as raw materials
(malts, adjuncts, and hops) and mashing. The FAs content of beer
In conclusion, a simple and reliable GC-FID method with liquid–
is also influenced by the process of maturation, during the matura-
liquid cartridge extraction and SPE purification was developed for
tion yeast growth determines an increased of biosynthesized FAs
the quantitative determination of FFAs in beer. The LLCE with
(Bravi, Perretti, et al., 2009). Moreover, the yeast strain and its
ChemElut cartridges and n-hexane/diethyl ether mixture allows
metabolism and catabolism influences the FAs in beer samples.
the efficient extraction (recovery between 78% and 104%) of
The EB and the BB showed the higher content of FFAs,
lipophilic analytes (medium and long chain fatty acids), in traces
11.638 mg/L and 9.382 mg/L respectively; probably the presence
in aqueous medium, from a difficult matrix as beer, that it is gen-
of crude cereals as adjunct influenced the lipid content of final
erally recognized in the literature for its dishomogeneity (liquid,
products. Moreover, different malt and hops with different FA con-
gas, solids). The performance criteria of the proposed method
tent, that influence the FA pattern of finished beer, were used.
demonstrated that it is useful for the analysis of a wide range of
The lower content of FFAs was observed for amber beer sample
FFAs in different beer samples at suitable levels. The applicative
(6.450 mg/L), the higher temperatures necessary to obtain more
analyses of FAs in experimental beers confirmed the validity of
coloured malt during malting process could probably reduce the
the developed method when applied on beer samples experimen-
lipid content of malted cereals.
tally brewed with different raw materials (malt, hop, and crude
In the analyzed beer samples, the most representative fatty
cereals) and different yeast, highlighting their influence on the
acids were the unsaturated oleic and linoleic acids, but also the
FAs pattern of final beer.
linolenic acid (not detectable only in EMB sample), and the satu-
rated palmitic and stearic acids.
For amber beer the more representative FA was linoleic References
(2.465 mg/L), followed by palmitic (0.981 mg/L), oleic (0.935 mg/
L), stearic (0.882 mg/L), and linolenic (0.814 mg/L) acids, Ahuja, S., & Scypinski, S. (2001). Handbook of modern pharmaceutical analysis
(pp. 415–443). New York: Academic Press.
respectively. For the crude wheat added BB, linoleic acid was the Anness, B. J. (1984). Lipids of barley, malt and adjuncts. Journal of the Institute of
major FA (3.714 mg/L), followed by linolenic (1.343 mg/L), oleic Brewing, 80, 315–318.
(1.325 mg/L), stearic (1.188 mg/L), palmitic (0.930 mg/L), and AOAC. (2002). Guidelines for single laboratory validation of chemical methods for
dietary supplements and botanicals.
lauric (0.495 mg/L) acids. This beer, added with wheat, showed AOAC International (2013). Definitions and calculations of Horrat values from
higher level of linoleic acid (principal component of wheat lipids) interlaboratory data. Accessed 1 October 2015
in comparison with all-malt beer (AB), this is probably due to the Bamforth, C. W. (1985). The foaming properties of beer. Journal of the Institute of
Brewing, 91, 370–383.
presence of crude wheat and to the fatty acid profile of its lipids
Battistutta, F., Buiatti, S., Zenarola, C., & Zironi, R. (1994). Rapid analysis of free
(Macmurray & Morrison, 1970; Welch, 1975). Oleic acid was the medium chain fatty acids and related ethyl esters in beer using SPE and HRGC.
dominant FA (2.372 mg/L) in EMB, followed by linoleic Journal of High Resolution Chromatography, 17, 662–664.
(1.880 mg/L), palmitic (1.819 mg/L), and stearic (1.685 mg/L) acids. Bravi, E., Benedetti, P., Marconi, O., & Perretti, G. (2014). Determination of free fatty
acids in beer wort. Food Chemistry, 151, 374–378.
The FA composition of T. dicoccum, showing an higher content of Bravi, E., Marconi, O., Perretti, G., & Fantozzi, P. (2012). Influence of barley variety
oleic acid compared with FA pattern of barley malt (Suchowilska and malting process on lipid content of malt. Food Chemistry, 135, 1112–1117.
346 E. Bravi et al. / Food Chemistry 215 (2017) 341–346

Bravi, E., Perretti, G., Buzzini, P., Della Sera, R., & Fantozzi, P. (2009). Technological Kobayashi, N., Kaneda, H., Kano, Y., & Koshino, S. (1993). Determination of fatty acid
steps and yeast biomass as factors affecting the lipid content of beer during the hydroperoxides produced during the production of wort. Journal of the Institute
brewing process. Journal of Agricultural and Food Chemistry, 57(14), 6279–6284. of Brewing, 99, 143–146.
Bravi, E., Perretti, G., Montanari, L., Favati, F., & Fantozzi, P. (2007). Supercritical fluid Macmurray, T. A., & Morrison, W. R. (1970). Composition of wheat-flour lipids.
extraction for quality control in beer industry. Journal Supercritical Fluids, 42, Journal of the Science of Food and Agriculture, 21, 489–544.
342–346. MacPherson, J. K., & Buckee, G. K. (1974). Estimation of free fatty acids (C8–C18) in
Bravi, E., Sensidoni, M., Floridi, S., & Perretti, G. (2009). Fatty acids composition in wort and beer. Journal of the Institute of Brewing, 80, 540–544.
beer lipids and determination of corn adjuncts. MBAA Technical Quarterly, 46(4). Mayer, H., Marconi, O., Perretti, G., Sensidoni, M., & Fantozzi, P. (2011). Investigation of the suitability of hulled wheats for malting and brewing. Journal of the
Carrington, R., Collett, R. C., Dunkin, I. R., & Halek, G. (1972). Gushing promoters and American Society of Brewing Chemists, 69(2), 116–120.
suppressants in beer and hops. Journal of the Institute of Brewing, 78(3), Meilgaard, M. C., Dalgliesh, C. E., & Clapperton, J. F. (1979). Beer flavour terminology.
243–254. Journal of the Institute of Brewing, 85, 38–42.
Chen, E. C.-H., Jamieson, A. M., & Van Gheluwe, G. (1980). The release of fatty acids Muller, M. P., Schmid, F., Becker, T., & Gastl, M. (2010). Impact of different hop
as a consequence of yeast autolysis. Journal of the American Society of Brewing compounds on the over foaming volume of beer caused by primary gushing.
Chemists, 38, 148–153. Journal of the Institute of Brewing, 116(4), 459–463.
Christian, M., Titze, J., & Ilberg, V. (2011). Chemical structure of model substances Ng, L. K. (2002). Analysis by gas chromatography/mass spectrometry of fatty acids
related to their gushing-inducing and -suppressing activity. Journal of the and esters in alcoholic beverages and tobaccos. Analytica Chimica Acta, 465,
American Society of Brewing Chemists, 69(3), 170–179. 309–318.
Clapperton, J. F. (1978). Fatty acids contributing to caprylic flavour in beer. The use Schutz, M., & Back, W. (2005). The influence of mash acidification on long-chain
of profile and threshold data in flavour research. Journal of the Institute of fatty acid content in wort determinate by a new developed method.
Brewing, 84, 107–112. Monatsschrift fur Brauwissenschaft, 58, 6–10.
DeVries, K. (1990). Determination of free fatty acids in wort and beer. Journal of the Segawa, S., Yamashita, S., Mitani, Y., & Masachika, T. (2002). Analysis of detrimental
American Society of Brewing Chemists, 48(1), 13–17. effect on head retention by low molecular weight surface-active substances
Dickie, K. H., Cann, C., Norman, E. C., Bamforth, C. W., & Muller, R. E. (2001). Foam- using surface excess. Journal of the American Society of Brewing Chemists, 60,
negative materials. Journal of the American Society of Brewing Chemists, 59(1), 31–36.
17–23. Shabir, G. A. (2003). Validation of high-performance liquid chromatography
Eurachem Working Group. (2014). Eurachem guide. The fitness for purpose of methods for pharmaceutical analysis: Understanding the differences and
analytical methods. A laboratory guide to method validation and related topics. similarities between validation requirements of the US Food and Drug
Middlesex, U.K. Administration, the US Pharmacopeia and the International Conference on
Food and Drug Administration, ORA Laboratory Procedure (2014). Methods, method Harmonization. Journal of Chromatography A, 987, 57–66.
verification and validation, laboratory manual, (Vol. II, Document number: Sistla, R., Tata, V. S. S. K., Kashyap, Y. V., Chandrasekar, D., & Diwan, P. V. (2005).
ORA-LAB.5.4.5). Accessed 12/10/2015 Journal of Pharmaceutical and Biomedical Analysis, 39, 517–522.
ScienceResearch/FieldScience/LaboratoryManual/UCM092147.pdf. Suchowilska, E., Wiwart, M., Borejszo, Z., Packa, D., Kandler, W., & Krska, R. (2009).
Hawthorne, D. B., Jones, R. D., Barrett, P. A., Kavanagh, T. E., & Clarke, B. J. (1986). Discriminant analysis of selected yield components and fatty acid composition
Methods for the analysis of C4 to C10 fatty acids in beer, wort and carbohydrate of chosen Triticum monococcum and Triticum dicoccum and Triticum spelta
syrup. Journal of the Institute of Brewing, 92, 181–184. accessions. Journal of Cereal Science, 49, 310–315.
Horak, T., Culik, J., Cejka, P., Jurkova, M., Kellner, V., Dvorak, J., et al. (2009). Analysis Taylor, G. T., & Kirsop, B. H. (1977). Rapid method for the analysis of the fatty acids
of free fatty acids in beer: Comparison of solid-phase extraction, solid-phase of fermenting wort and beer (C6–C10) and yeast (C8–C18). Journal of the
microextraction, and stir bar sorptive extraction. Journal of Agricultural and Food Institute of Brewing, 83, 97–99.
Chemistry, 27, 11081–11085. Van der Meersche, J., Devreux, A., & Masschelein, C. A. (1979). Formation des acides
Horak, T., Culik, J., Jurkova, M., Cejka, P., & Kellner, V. (2008). Determination of free volatils dans la maturation de la biere. In Proceedings of the European Brewery
medium-chain fatty acids in beer by stir bar sorptive extraction. Journal of Convention congress, Berlin (pp. 787–800). Zoeterwoude, The Netherlands: EBC.
Chromatography A, 1196–1197, 96–99. Vanderhaegen, B., Nevin, H., Verachtert, H., & Derdelinckx, G. (2006). The chemistry
Irwin, A. J., & Thompson, D. J. (1987). A rapid method for the extraction and analysis of beer aging – A critical review. Food Chemistry, 95, 357–381.
of beer flavour components. Journal of the Institute of brewing, 93, 113–115. Welch, R. (1975). Fatty acid composition of grain from winter and spring sown oats,
Kaneda, H., Kano, Y., Kamimura, M., Osawa, T., & Kawakishi, S. (1990). Analysis of barley and wheat. Journal of the Science of Food and Agriculture, 26, 429–435.
long-chain fatty acids in beer by HPLC-fluorescence detection method. Journal of
Agricultural and Food Chemistry, 38, 1363–1367.