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Article
Characterization of the Migration of Hop Volatiles into Different Crown Cork Liner Polymers and Can Coatings Philip Ché Wietstock, Richard Glattfelder, Leif-Alexander Garbe, and Frank-Jürgen Methner J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00031 • Publication Date (Web): 20 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016
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Journal of Agricultural and Food Chemistry
Revised manuscript ID jf-2016-00031j 1.
Title: Characterization of the Migration of Hop Volatiles into Different Crown Cork Liner Polymers and Can Coatings.
2.
Corresponding author: Dipl.-Ing. Philip C. Wietstock, Technische Universität Berlin, Department of Food Technology and Food Chemistry, Chair of Brewing Science, Seestrasse 13, 13353 Berlin, Germany,
[email protected].
3.
–
4.
Coauthors list: B.Sc. Richard Glattfelder, Technische Universität Berlin, Department of Food Technology and Food Chemistry, Chair of Brewing Science, Seestrasse 13, 13353 Berlin, Germany,
[email protected] Prof. Dr. Leif-Alexander Garbe, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Hochschule Neubrandenburg, Brodaer Str. 2 D, 17033, Neubrandenburg, Germany,
[email protected] Prof. Dr.-Ing. Frank Jürgen Methner, Technische Universität Berlin, Department of Food Technology and Food Chemistry, Chair of Brewing Science, Seestrasse 13, 13353 Berlin, Germany,
[email protected].
5.
Significance: Scalping of volatile hop aroma compounds by crown cork liner polymers and by beer can coatings was characterized in buffered model systems and in beer using solvent assisted flavor evaporation (SAFE)GC/MS. The work provides new information regarding the degree of scalping and the rate by which scalping of hop volatiles occurs. Migration of certain hop aroma compounds was shown to be strongly dependent on the polaritiy of the hop volatile measured and to a lesser extent on the closure or packaging used. A short exposure time to the packaging material already yielded a high migration of certain compounds which alters the original balance of the flavor compounds and thus results in flavor loss and flavor deterioration. This is the first time that the scalping of hop volatiles in beer systems was described by Fick’s second law of diffusion. Outcomes from this study point to the importance of scalping in beer systems and will therefore help improving closures and to ‘rethink’ experimental designs when assessing hop aroma.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Characterization of the Migration of Hop Volatiles into Different Crown Cork Liner Polymers and Can Coatings. Philip C. Wietstock1*, Richard Glattfelder1, Leif-Alexander Garbe2, Frank-Jürgen Methner1
1
Technische Universität Berlin, Department of Food Technology and Food Chemistry, Chair
of Brewing Science, Seestrasse 13, 13353 Berlin, Germany. 2
Hochschule Neubrandenburg, Department of Agriculture and Food Sciences, Brodaer Str. 2,
17033 Neubrandenburg, Germany.
* Corresponding author: Philip C. Wietstock, Seestr. 13, D-13353 Berlin, Phone: +4930-31427505, Fax: +493031427503, Email:
[email protected] 2 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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ABSTRACT
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Absorption of hop volatiles by crown cork liner polymers and can coatings was investigated
3
in beer during storage. All hop volatiles measured were prone to migrate into the closures and
4
the absorption kinetic was demonstrated to fit well Fick’s 2nd law of diffusion for a plane
5
sheet. The extent and rate of diffusion was significantly dissimilar and was greatly dependent
6
on the nature of the volatile. Diffusion coefficients in cm2/day ranged from 1.32×e-5
7
(limonene) to 0.26×e-5 (α-humulene). The maximum amounts absorbed into the material at
8
equilibrium were in the order limonene > α-humulene > t-caryophyllene > myrcene >>
9
linalool > α-terpineol > geraniol. Applying low-density polyethylene (LDPE) liners with
10
oxygen scavenging functionality, oxygen barrier liners made up from high-density
11
polyethylene (HDPE), or liner polymers from a different manufacturer had no significant
12
effect on the composition of hop volatiles in beers after prolonged storage of 55 days;
13
however, significantly higher amounts of myrcene and limonene were found in the oxygen
14
barrier-type crown cork while all other closures behaved similarly. Can coatings were
15
demonstrated to absorb hop volatiles in a similar pattern as crown corks but to a lesser extent.
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Consequently, significantly higher percentages of myrcene were found in the beers.
17 18
Keywords: Scalping, crown cork liner polymers, hop volatiles, can coatings, hop aroma.
3 ACS Paragon Plus Environment
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INTRODUCTION
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Flavor scalping is referred to as the loss of aroma substances in food containers due to
21
migration into the packaging materials. This process can occur in both directions and product
22
volatiles can either migrate into the packaging or or substances from the packaging material
23
can migrate into the product as e.g. bisphenols do in canned beers1,2, or chloranisoles3,4 in
24
wine . Most knowledge about flavor sorption today originates from the food industry, where
25
aroma migration of volatiles from juices or other citrus fruit containing beverages and
26
foodstuffs were studied.5,6 But also wine closures were studied intensively and were shown to
27
have a significant impact on the quality of the product.7,8 The sorption kinetics for these
28
reactions are from great interest because they allow deductions for the rate at which sorption
29
occurs, give indication of over-all rate of uptake, and open of a large field of modeling
30
complex applications.
31
A wide range of plastics are nowadays used for food packaging including homo-polymers or
32
co-polymers from low to high density polyethylene. Preferably polyethylene (PE) and
33
polypropylenes (PP) are used if plastics are in contact with foodstuffs because of their good
34
chemical resistance, inertness to most foods, good barrier properties to water, and
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thermostability.9 To minimize oxygen permeation, thus increasing shelf-life and organoleptic
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properties of the product, additional scavenging or barrier material may be introduced into the
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compound polymer. The extent of flavor absorption is influenced by the chemical
38
composition of the polymer, the molecular properties of the aroma compounds and external
39
factors.10-12
40
Today more than 400 single hop oil components are identified in hops and discovery of new
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compounds is still ongoing.13 The most abundant fractions are the hydrocarbons which are
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constituted of small isoprene units forming the monoterpenes or sesquiterpenes. In fresh hops,
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myrcene makes up to 69 %14 of the total hop oil composition and it has been reported by 4 ACS Paragon Plus Environment
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Schieberle and Steinhaus15 to be one of the most potent hop odorants. The terpenoids or
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terpene alcohols are oxygenated terpenes which feature a much higher solubility in beer.16
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Terpenoids may either be formed via oxidation of their corresponding terpenes17, are
47
enzymatically converted18,19 or released.20 Major components of the terpenoid fraction are
48
linalool, geraniol, and α-terpineol, all of which are believed to be an important contributor for
49
a floral hop aroma.16,21 The group of the oxygenated sesquiterpenes are claimed to be the
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source of the traditional spicy and herbal ‘noble-hop’ or ‘kettle-hop’ aroma.22 Substances
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frequently associated with these aromas are oxidation products of α-humulene and t-
52
caryophyllene.23 Furthermore, recent research focused on sulfur-containing compounds in hop
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oils which are present at much lower concentrations than the other fractions but concomitantly
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have very low flavor thresholds.16
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Hop volatiles in beer usually are rather low in concentration which makes it particularly
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difficult to quantitate those substances. The volatile nature of essential oils enables relatively
57
simple enrichment and separation using steam distillation, as described in ASBC24 and EBC25
58
manuals or the simultaneous extraction distillation (SED) described by Likens and
59
Nickerson.26 Solid-phase micro-extraction (SPME) coupled with a suitable measuring devices
60
and detectors such mass spectrometers (MS) are used nowadays by many laboratories.27-29
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Purge-and-trap30, head-space trap31, or thermal desorption32 are further alternatives.
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To achieve a beer rich in hop aroma, hops are added later on during whirlpool rest or during
63
late boil, in the cold part of the brewery mostly during secondary fermentation or maturation,
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or even in the form of distinct hop oil rich products downstream. Once the beer is bottled, the
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hop aroma changes rapidly during storage and only little is known about the origin of these
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flavor alterations. While storage conditions such as e.g. temperature certainly have a strong
67
impact on the rate and type of flavor changes in the product, closures also play a key role
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because they not only determine oxygen exposure of the product but moreover scalping of 5 ACS Paragon Plus Environment
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flavor compounds as Peacock and Deinzer11 indicated. However, also racemization33, acid-
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catalyzed isomerization34-36 or acid hydrolysis11 may explain the transitory nature of hop
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aroma in bottled beer. Only little work on aroma scalping and sorption kinetics in beer
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systems is available thus far. Peacock and Deinzer11 studied scalping of crown liners
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containing polyvinylchloride (PVC) and found particularly high absorptions of hydrocarbons
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along with varying extents of other beer constituents, not only those derived from hops. PVC
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features great flexibility when used in combination with plasticizers such as phthalates but
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because of safety concerns is today mainly used in nonfood packaging applications.37 In 2013,
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Van Opstaele et al.38 reported a decrease of hop volatiles in beer and ascribed it partly to
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scalping into crown cork liners but also to adsorption to haze formed during ageing. The
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composition and material of the crown corks used in this study were not mentioned, though.
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Information as related to absorption of aroma-active beer constituents by different ‘modern’
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PE beer bottle closures and can coatings as well as the concurrent uptake-rate of hop volatiles
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into crown cork liners is thus far lacking in published literature. The goal of this study was
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consequently to elucidate these issues. Hence, for the first time, time-dependent uptake rates
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and diffusion coefficients for a hop volatile-LDPE liner system in beer during storage were
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deduced using Fick’s 2nd law of diffusion. Additionally, the effects of using liners from
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different manufacturers, crown corks with oxygen scavenging or oxygen barrier functionality,
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or storing beers in cans on hop volatile concentration in PE liner polymers, can linings, and
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corresponding beers after prolonged storage were analyzed. Solvent-assisted flavor
89
evaporation (SAFE) coupled with GC/MS was used to quantitate hop volatiles in beers or
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packaging because this procedure ensures a gentle sample treatment and an appropriate
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detection of the target analytes.39 This work provides novel information regarding the extent
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of scalping of hop volatiles in beer systems and the rate by which scalping occurs. Outcomes
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from this study therefore allow deductions on the loss of certain volatiles to the closures or 6 ACS Paragon Plus Environment
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packaging tested and therefore point to a disturbance of the original balance between beer
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aroma compounds eventually altering the beer’s aroma perception.
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7 ACS Paragon Plus Environment
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MATERIALS AND METHODS
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Chemicals.
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Acetic acid, geraniol, α-humulene, limonene, linalool, myrcene, 4-octanol, and trans-
100
caryophyllene were obtained from Sigma-Aldrich (Steinheim, Germany). Ethanol, sodium
101
acetate, sodium carbonate, and sodium sulphate were ordered from Merck (Darmstadt,
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Germany). Diethyl ether was obtained from VWR (Leuven, Belgium). All chemicals were of
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analytical grade or highest possible purity.
104 105
Beer analysis according to MEBAK.40
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The beer analyses carried out were as follows: original extract (2.9.6.3); alcohol (2.9.6.3); pH-
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value (2.13); color (2.12.2).
108 109
Quantitation of hop volatiles in beer.
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Hop volatiles were quantitated following a modified procedure described by Engel, Bahr and
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Schieberle.39 This procedure uses the solvent assisted flavor evaporation (SAFE) technique
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and GC combined with MS analysis. Beer samples were filtered (funnel and filter sheet) to
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remove CO2 and an aliquot of 100 mL was spiked with 4-octanol such that a 1 mL addition
114
achieved a final concentration of 25 µg/L in the sample. The sample was subsequently
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extracted twice with 150 mL diethyl ether and placed into a SAFE-apparatus. After high
116
vacuum distillation, the sample was washed twice with a 0.5 M Na2CO3 solution and
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bidistilled water, then dried over Na2SO4 for one hour, and concentrated to 5 mL using a
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vigreux column at a temperature of 46-48 °C. A sample (1 µL) of the concentrated distillate
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was introduced into the GC via a cold injection system (Gerstel GmbH & Co. KG, Mühlheim
120
an der Ruhr, Germany) in 20:1 split mode to a GC 6890 (Agilent Technologies, Waldbronn,
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Germany) fitted with a capillary column (VF-5 MS, 60 m x 0.25 mm, 0.25 µm film, Varian, 8 ACS Paragon Plus Environment
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Darmstadt, Germany). The following temperature program was used for the GC oven: after 12
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min at 35 °C, the oven temperature was raised to 150 °C at a rate of 12 °C/min and then to
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250 °C at 30 °C/min where it was held for 5 min. The flow rate of helium carrier gas was 0.6
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mL/min. Mass spectra in the electron impact mode (MS/EI) were generated at 70 eV using an
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MSD 5973 (Agilent Technologies, Waldbronn, Germany) in selected ion monitoring (SIM)
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mode. The analytes’ retention times and m/z ratios are depicted in Table 1. A six-point
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external calibration (r2 > 0.99) was carried out to quantitate the target molecules. The
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method’s limit of quantitation (LOQ) was 1 µg/L for all analytes. The calculated
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concentrations in µg/L were divided by the volume of the initial sample to obtain the total
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amounts of analytes in the samples.
132 133
Quantitation of hop volatiles in crown cork liner polymers and can linings.
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Crown corks with liners were removed from buffer solutions or beer bottles, washed three
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times with 12 mL of bidistilled water, and any liquid residues were carefully dried off with a
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tissue. Crown cork liner polymers were then extracted with 100 mL of diethylether over 24
137
hours at room temperature and an amount of 4-octanol was added as an internal standard to
138
achieve a final concentration of 25 µg/L in the sample. Beer cans were cut into small pieces
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and were subsequently washed with 800 mL of bidistilled water. The extraction procedure
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was then analogous to the crown liner extraction. The diethylether extracts from crown liner
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materials, beer cans, or water were then analyzed as described above. There is a possibility
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that analytes are removed by water during the washing step. The potential washing-off effect
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was therefore tested by adding 200 mL of water to already rinsed can material and shaking it
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for 5 min vigorously. The water was then separated from the can material and extracted twice
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with 150 mL diethylether prior to analyzing it as described above.
146 9 ACS Paragon Plus Environment
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Migration of hop volatiles into crown liner materials in buffered model solutions.
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A preliminary experiment was conducted to assess principal migration of hop volatiles in
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crown cork liner polymers. Low-density polyethylene crown corks were therefore incubated
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for 24 and 72 hours in 130 mL of buffered model solutions (acetate buffer, 0.01 M, pH 4.3,
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5 vol.-% EtOH) which was spiked with 130 µg of myrcene, limonene, linalool, α-terpineol,
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geraniol, t-caryophyllene, and α-humulene prior to adding the crown corks. The incubation
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flasks were filled with minimized headspace in order to minimize the diffusion of hop
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volatiles into the gaseous phase. The amount of volatiles in the liner polymer was then
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analyzed as described above.
156 157
Aroma sorption kinetics during beer storage.
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30 L of a commercially available pilsner beer (original gravity: 11.84 % wt./wt.; alc.: 5.2 %
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v/v; bitter units: 28; carbon dioxide: 5.1 g/L; pH: 4.38, color: 6.38 °EBC; hop dosage solely
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CO2 extract at the beginning of wort boiling; all values were measured according to
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MEBAK40) low in hop aroma (all hop constituents < 5 µg/L as measured by SAFE-GC/MS)
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was spiked with a mixture of myrcene, limonene, linalool, α-terpineol, geraniol, t-
163
caryophyllene, and α-humulene such that a final amount in the beer of 250 µg for each
164
compound was achieved. All compounds were solved in 10 mL of ethanol prior to spiking
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directly into the keg. The keg remained open as short as possible during spiking to minimize
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oxygen uptake. The beer was mixed well and was immediately filled in 0.5 L standard brown
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glass bottles using a H4 filler (JS Maschinen GmbH, Nandlstadt, Germany). The bottles were
168
pre-flushed three times with CO2 prior to filling and were directly sealed using low-density
169
polyethylene (LDPE) pry-off crown corks. Oxygen levels in packaged beer after filling were
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< 50 µg/L as measured using a DIGOX 6.1 (Dr. Thiedig GmbH & Co KG, Berlin, Germany)
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apparatus. During storage at room temperature, the spiked beers’ amounts of hop volatiles 10 ACS Paragon Plus Environment
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were measured directly after filling, after 21 days, and after 55 days of storage, respectively.
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The crown cork liner polymers were analyzed after 0, 1, 3, 8, 21, and 55 days of storage. Each
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specified day, three bottles were taken out of the storage experiment and were analyzed
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separately. The aroma sorption kinetics were modeled additionally using eq. 1:
176 177
Mt 8 = 1− 2 M∞ π
n =∞
1
∑ (2n + 1)
2
{
exp − D ( 2n + 1) 2 π 2 t / l 2
}
(1)
n =0
178
where: Mt is the total amount of components absorbed by the sheet at time t, expressed as µg
179
of aroma component, M∞ the equilibrium absorbed mass which is theoretically attained after
180
infinite time, expressed as µg of aroma component, D is the diffusion coefficient, expressed
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as cm2/day, and l is the film thickness of the polymer, expressed as cm.
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Eq. 1 is derived from Fick’s 2nd law.41 The application of eq. 1 is based on the assumption of
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an instantaneous pressure change of sorbing compounds above a homogenous thin film made
184
of a dense polymer which remains constant afterwards. For simplification, the crown liner
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material was assumed to be a plane sheet which does not swell and the polymer was
186
considered to be initially free of sorbing compounds. Assuming the diffusion coefficient to be
187
constant did not yield satisfactory results because clearly, the diffusion coefficient is
188
concentration dependent and increases as the concentration of the migrating molecule
189
increases. The concentration-dependent diffusion coefficients Dc were therefore deduced
190
separately from observations of the time-dependent rate of uptake of the individual hop
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volatiles into the liner material. Final D values given represent then the mean of the variable
192
diffusion coefficients averaged over the range of concentration. In the case of t-caryophyllene
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and α-humulene, the equilibrium sorption was not reached during the experiment and their
194
mean diffusion coefficients could thus not be derived. Therefore, their diffusion coefficients
195
were approximated by non-linear fitting of the experimental data to eq. 1. 11 ACS Paragon Plus Environment
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Characterization of aroma migration in different liner polymers.
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The same beer and sample preparation as in the previous trial was applied but in this trial four
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different pry-off crown cork liner materials were used: ‘standard’ LDPE liner from a
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manufacturer A, ‘standard’ LDPE liner from a manufacturer B, oxygen barrier high-density
200
polyethylene (HDPE) liner, and oxygen scavenger LDPE liner with Na2SO3 as the reactant in
201
the liner material. The bottles were stored for 55 days at room temperature and three crown
202
corks or three bottles, respectively, were analyzed individually on their amounts of hop
203
volatiles.
204 205
Comparison of hop volatile migration into crown cork liner materials and can coatings.
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A highly-hopped ale-type beer (dry-hopped, 5.6 vol.-% EtOH) from Scotland was available in
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both 0.33 L cans and 0.33 L bottles (capped with pry-off LDPE crown cork liners with
208
oxygen scavenging functionality). It was ensured that the beers were from the same
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maturation tank. Storage was then conducted in the dark at room temperature for 46 days and
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hop volatiles in beers and packaging materials were subsequently quantitated by SAFE-
211
GC/MS from three individual samples.
212 213
Data Analysis.
214
Statistical analysis was performed using XLSTAT (Ver. 2014.5.03.), Addinsoft, USA. Data
215
were analyzed using analysis of variance (ANOVA). Means were used to compare differences
216
and Tukey’s honestly significant difference (HSD) test was applied to compare the mean
217
values. The significance level for the ANOVA analyses was p < 0.05. Additionally, an
218
unpaired Student’s t-test was used to compare sample means between beers filled in cans and
219
filled in bottles at a confidence level of 95 %.
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RESULTS AND DISCUSSION
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A bottle or canned beer can be considered a system comprising gaseous and liquid phases.
222
Directly after filling and sealing, the system is not in equilibrium and molecules strive from
223
the liquid phase into the gaseous phase or backwards. The compounds’ distribution in both
224
phases at equilibrium is defined by their distribution factor kL,G42 (eq. 2) and by the system’s
225
conditions such as e.g. the temperature:
226
k L ,G =
227
where: kL,G is the (thermal) distribution factor between the liquid and the gaseous phase, cG is
228
the molar concentration of a component in the gas phase, and cL is the molar concentration of
229
a component in the liquid phase.
cG cL
(2)
230 231
If scalping occurs, e.g. in beer bottles, this system is disturbed because molecules migrate
232
from the gaseous phase or headspace into the packaging material or crown cork liner material
233
thus lowering the concentration in the gaseous phase. The over-all migration in the packaging
234
is defined by a second partition coefficient kG,P between the product side; here, the gaseous
235
phase or headspace, and the packaging’s polymer material at equilibrium (eq. 3).9
236
kG ,P =
237
where: kG,P is the partition coefficient between the headspace and the packaging polymer, cP is
238
the concentration of a component in the packaging polymer, and cG is the concentration of a
239
component in the gaseous phase or headspace.
cP cG
(3)
240 241
When migration into packaging polymers occurs, the component’s concentration in the
242
gaseous phase is lowered and consequently, molecules strive again for equilibrium and diffuse
243
from the liquid phase into the gaseous phase as defined by their kL,G value. If a component is 13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
244
volatile (kL,G > 1), the limiting factor for the over-all rate of a component sorbing into the
245
packaging must therefore be kG,P because the headspace’s ‘reservoir’ is always ‘filled up’
246
again; at least as long as the component is not completely exhausted in the liquid.
247
However, it shouldn’t be neglected that the packaging material is not a closed system but is
248
‘connected’ to the environment, and also permeation from the packaging into the environment
249
may occur. Ultimately, aroma is lost and/or is altered continuously, not only because aromatic
250
molecules migrate into the packaging material via the headspace but also because the
251
equilibrium state is never reached when permeation occurs. Within this study’s scope, the
252
determination of kG,P was not possible because the component’s kL,G values were not known
253
and concentrations in the headspace were not measured. However, this information is still
254
essential to understand and interpret this study’s results.
255 256
Migration of hop volatiles from buffered model solutions into LDPE crown cork liner
257
materials.
258
In a first test series of this study, LDPE crown cork liners without any barrier or scavenging
259
materials added to the liner polymer were screened on their tendency to scalp a selection of
260
hop volatiles. Additional to investigating the liner materials’ scalping tendencies, it was tested
261
if the 24 hour extraction procedure via diethylether was sufficient to extract the analytes from
262
the liner material. As analytical markers, two monoterpenes (myrcene, limonene), two
263
sesquiterpenes (α-humulene, t-caryophyllene), and three monoterpenoids (linalool, geraniol,
264
and α-terpineol) commonly found in beer and hops were selected because they not only are
265
odor-active constituents in beer but also represent different chemical characteristics, e.g.
266
polarities.
267
In individual trials, crown corks were incubated for 24 hours or 72 hours in ‘beer-like’
268
buffered model solutions which were spiked with hop volatiles prior to adding the caps. A 24 14 ACS Paragon Plus Environment
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hour extraction with diethylether and subsequent SAFE-GC/MS analysis revealed that in
270
terms of the hydrocarbon fraction 8.4 µg myrcene, 9.1 µg limonene, 7 µg t-caryophyllene,
271
and 7.7 µg α-humulene were absorbed by the liner polymer. The migration of terpenoids was
272
lesser and only 2 µg of linalool, 1.6 µg of α-terpineol, and 2.6 µg of geraniol were found
273
(Table 2). Incubating the crown corks for 72 hours at the same experimental conditions
274
yielded a further extraction of maximum 4.5 % for the hydrocarbon fraction while the
275
terpenoid fraction showed only very little changes (< 0.3 µg).
276
A second diethylether extraction of the same crown cork liner materials for 24 hours in
277
diethylether yielded very minor amounts of hydrocarbons (< 0.1 µg) while no further
278
terpenoids were detected in the concentrated extracts. The extraction for 24 hours was
279
therefore considered sufficient and was used for all the trials.
280 281
Hop volatile sorption into an LDPE liner polymer as a function of storage time.
282
A storage trial was prepared in which a commercial Pilsner type beer was spiked with 250 µg
283
of hop volatiles per 500 mL of beer. The beers were capped with ‘standard’ LDPE crown
284
caps, and the beers’ and liners’ content of hop volatiles were measured at certain time points.
285
Figure 1 depicts the content of hop compounds as detected in the liner materials directly after
286
bottling, as well as after 1, 3, 8, 21 and 55 days of storage. The drawn through lines in the
287
figure were obtained by applying eq. (1) to the data. Already after one day of storage, hop
288
volatiles were present in the liner material in contents ranging from < 0.5 µg (geraniol) to
289
40.7 µg (limonene). Until day 21, a distinct increase was observed which leveled off for
290
myrcene as well as limonene, while a further increase was seen for t-caryophyllene and α-
291
humulene. Linalool, geraniol, and α-terpineol showed a similar behavior and curve shape as
292
myrcene and limonene but at a much lower rate. From all substances measured, from initially
293
250 µg spiked amount in the bottles, the following order of sorption into the LDPE-liner 15 ACS Paragon Plus Environment
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polymer from highest to lowest was found: limonene (105.7 %) > α-humulene (91.4 %) > t-
295
caryophyllene (74.2 %) > myrcene (53.1 %) >> linalool (1.4 %) > α-terpineol (0.6 %) >
296
geraniol (0.4 %).
297 298
Directly after spiking, the rates of terpenes and sesquiterpenes found in the beers were low
299
and ranged from 10.9 % (myrcene) to 20.4 % (t-caryophyllene) as related to their initially
300
spiked amount while the terpene alcohols’ rates were higher and ranged from 71.9 %
301
(linalool) to 81.7 % (α-terpineol) (Figure 1). The gaseous phase is not obtained analytically
302
when doing a liquid-liquid extraction which is the first step when doing the SAFE-analysis.
303
One reason for the low amounts of terpenes and sesquiterpenes detected after the initial
304
spiking may be that they accumulated in the gaseous phase or bottle headspace as based on
305
their individual kL,G value and therefore were not extracted when performing the diethylether
306
extraction process. Myrcene is less polar than e.g. linalool because of its more aliphatic
307
character. Therefore, it presumably has a higher kL,G value in an aqueous system (e.g. beer)
308
than linalool and thus, at equilibrium, myrcene molecules are present to a higher extent in the
309
bottle headspace than e.g. linalool molecules. Consequently, less myrcene is detected when
310
doing the liquid-liquid extraction.
311
This hypothesis is supported by the observation that all more polar compounds measured with
312
a concurrent lower kL,G value and higher solubility in beer such as linalool, geraniol, and α-
313
terpineol were found to a higher extent in the beer after spiking while all hop constituents
314
measured with a more aliphatic character and concomitantly lower kL,G were detected at lower
315
levels. Unfortunately, no data is available on the distribution coefficients of hop volatiles in
316
beer systems which would allow predicting their concentration in the headspace. Linalool is
317
the only compound where little is known and in wort, a kL,G-value of 37.2 at a temperature of
318
72 °C was determined.42 The kL,G-values are dependent on the temperature, the pressure and 16 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
319
the composition of the gasous and liquid phases.42 In beer at room temperature, the
320
compounds’ distribution coefficients (kL,G) are presumably lower because of the lower
321
temperature and the beers’ ethanol content; however, they must still be greater 1 because they
322
are considered to be volatiles.
323
Adding up the amounts found in the crown cork liner materials and the beers after the 55 days
324
of storage revealed that the terpenes’ ‘total’ amounts increased over time, and in sum, the
325
following rates as related to the initial spiking amount were detected at the end of storage:
326
myrcene, 56.6 %; limonene, 114 %; t-caryophyllene, 78.9 %; α-humulene, 96.4 %.
327
Concomitantly, the terpene alcohols’ rates increased or decreased only marginally (linalool,
328
74.3 %; α-terpineol, 73.6 %; geraniol, 69.9 %). The increase of the terpenes’ or
329
sesquiterpenes’ ‘total’ content found in liner and beer over storage time may again be traced
330
back to and support the hypothesis given before: the terpenes’ or sesquiterpenes’ initially
331
accumulated in the bottle headspace at an amount as defined by their kL,G value directly after
332
bottling, and from there, subsequent slow diffusion into the crown cork liner material
333
occurred during storage as defined by their diffusion coefficients until an equilibrium was
334
reached.
335
This hypothesis, however, cannot fully explain the observations, and in addition, the low rates
336
detected in liner and beer, in particular for myrcene where still ‘only’ 56.6 % were found at
337
the end of storage, may also be originated from losses of analytes during sample preparation
338
such as e.g. during the filtration step of the beers before the diethylether extraction. Also,
339
losses of analytes during the bottling process cannot be fully excluded.
340
Ultimately, sorption in the liner materials results consequently in a lowering of the
341
concentrations in the headspace which, in turn, provokes that volatiles diffuse yet again in the
342
headspace striving for equilibrium. The observation that the terpenes and sesquiterpenes
343
showed reciprocal curve shapes in liner and beer over storage is therefore an indication that 17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
344
the scalping in fact is the cause for their depletion in the beers. Linalool, geraniol, and α-
345
terpineol, however, showed only little accumulations in the liner material and their
346
concentration changes during beer storage thus must be derived from other reactions. Linalool
347
is known to be relatively stable over storage which is in accordance to the results from this
348
study; however, there is evidence that racemization from the (R)- into the less aroma-active
349
(S)-enantiomer can occur.43 Geraniol and α-terpineol are reported to undergo certain
350
isomerization reactions34-36 and their decrease or increase, respectively, may thus be
351
originated from those transformations.
352 353
The absorption kinetic was modeled by fitting Fick’s second law (eq. 1) to the experimental
354
data. Table 3 lists the model parameters as calculated from eq. 1. The ratios between RMSE
355
and Mmax ranged from 2.3 to 19.1 % which was judged satisfactory. The substances t-
356
caryophyllene and α-humulene did not reach the equilibrium state during the 55 day storage
357
and the diffusion coefficients were therefore derived from fitting eq. 1 to the experimental
358
data by using non-linear regression analysis. ANOVA analysis of the results at p < 0.05
359
indicated a significant difference between the diffusion coefficients and a clustering into four
360
groups. The clustering as related to the substances implies that the different kinetics in
361
diffusing into the LDPE-film may be derived from the different chemical properties of the
362
volatiles such as most probably the polarity and the molecular size. The different Mmax values
363
observed can also only be partly ascribed to the different diffusion kinetics but are derived
364
from the compounds’ different affinities to react with the LDPE-polymer. The ‘polar’ terpene
365
alcohols linalool, α-terpineol, and geraniol clearly formed one group according to Tukey’s
366
HSD, while myrcene, limonene, α-humulene, and t-caryophyllene did not.
367
Taken these data together, the results suggest that all substances measured diffuse into the
368
LDPE-crown liner material according to Fick’s law which was also reported by other authors 18 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
369
for e.g. limonene44-46 or linalool.7,47 However, diffusion coefficients for the limonene-LDPE
370
system as reported in 46 or linalool-LDPE system as reported in 7 differed markedly from our
371
results which can most probably be traced back to the difference in the experimental
372
conditions used.
373 374
Hop volatile scalping characteristics of different crown cork liners.
375
In a separate trial, the migration of hop volatiles into four different crown cork liners and the
376
effect of their functionalities on the composition of hop volatiles in beer were tested. In
377
addition to the LDPE crown cork liner from manufacturer A, which was also used in the trial
378
before, a second LDPE crown cork liner from another manufacturer (manufacturer B), an
379
LDPE oxygen scavenging crown cork, and an HDPE oxygen barrier crown cork liner, both
380
again from manufacturer A, were examined. The amounts found in the different liners after 55
381
days of storage are shown in Figure 2.
382
As expected, the same scalping characteristics of the liner materials as in the previous trial
383
were observed, and hop terpenes and sesquiterpenes were found at high levels while the more
384
‘polar’ terpene alcohols were only present at very low levels. Interestingly, all liners displayed
385
similar scalping tendencies with the exception of the oxygen barrier liner where myrcene and
386
limonene were found at significantly higher levels. These results are contradicting findings
387
from 6 or 46 who reported that HDPE generally absorbs constituents to a lesser extent.
388
In the beers, none of the applied crown cork liner materials yielded significantly different
389
amounts of the constituents (Figure 3), and in accordance with the previous trial, the terpene
390
alcohols linalool, α-terpineol, and geraniol were found at elevated levels ranging from 140
391
(geraniol) to 182 µg (α-terpineol) while terpenes and sesquiterpenes were lower and ranged
392
from 7 (myrcene) to 15 µg (limonene).
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
393
As related to this, it should be noted that the oxygen barrier crown cork scalped significantly
394
higher amounts of myrcene and limonene but still no significant differences were found in the
395
beers. In this regard, also, the ‘total’ rate for limonene was found to be 149.8 % for the
396
oxygen barrier crown cork and it was therefore suspected that limonene was already present
397
in the HDPE liner material. Though, no contamination of the crown corks with the analytes
398
was supposed, all crown corks were still extracted individually and were tested on the
399
potential presence of hop volatiles in the liner materials to exclude this possibility. Yet, the
400
analysis revealed that none of the analytes were discovered at detectable levels in the liners.
401
The high recovery rates for limonene can therefore not be explained.
402
The crown corks’ oxygen scavenging and the oxygen barrier functions are assumed to
403
diminish the effects of oxygen in the bottled beer by binding oxygen which is already present
404
in the bottles after filling or by forming a physical barrier for oxygen entry through diffusion,
405
respectively. There is evidence that myrcene is readily oxidized when oxygen is present17 and
406
there is a possibility for the oxidation of myrcene in bottled beer. Yet, the observation that
407
both, the oxygen scavenger crown cork and the oxygen barrier crown cork, did not yield
408
significantly different amounts of hop volatiles in the bottled beers suggests that oxygen only
409
plays an inferior role for the changes of the hop volatile composition in beer after bottling,
410
though. It should however be emphasized that this outcome is only valid for this study’s scope
411
such as the limited storage time of 55 days and the constituents tested. For instance,
412
humuluene diepoxides are claimed to be important contributors to hop aroma48 which can be
413
lost by oxidation or acid hydrolysis.11 As discussed earlier, it is possible that hop volatiles are
414
present at high levels in the bottle headspace because of their volatility. One explanation for
415
the higher amounts of myrcene found in the oxygen barrier liner materials may therefore be
416
that the oxidation of myrcene as present in bottle the headspace was diminished because
417
oxygen diffusion through the crown cork liner was minimized thus yielding more myrcene to 20 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
418
migrate into the liner material. Aside from all that, diminishing oxygen exposure of beer or
419
other food systems is indispensable for preserving product freshness as it is also involved in
420
many flavor deterioration reactions.49,50
421 422
Scalping of hop volatiles in crown cork liner materials and can coatings.
423
The extent of flavor migration of hop volatiles into crown liner polymer and can coating
424
material was also investigated and beer from the same bright beer tank was filled in bottles or
425
cans prior to storing it for 46 days. The results from this trial are depicted in Table 4. In all
426
beers and liner materials or can coatings, low levels of limonene, t-caryophyllene, and α-
427
humulene were observed which may be traced back to the hop bill used for beer production. It
428
is therefore difficult to draw conclusions for these constituents. However, data still suggests
429
that the scalping was in accordance with the previous trials for crown cork liner materials.
430
Hop mono terpenes and sesquiterpenes were found at high levels in the crown cork liner
431
materials and high losses were thus detected in the beers. The terpene alcohols reacted
432
inversely and only small losses to the packaging material were observed. With regards to the
433
respective percentages of hop constituents scalped (Table 4), the can coatings were found to
434
be also liable to absorb hop mono terpenes and sesquiterpenes but exhibited a significantly
435
lower migration of myrcene (p < 0.05) into the packaging than the LDPE-polymer of the
436
crown corks. Trans-caryophyllene formed an exception and was only detected in the
437
packaging of both, LDPE-liners and can coatings while no t-caryophyllene was found in the
438
beers. Relative to the total amounts found in polymers and beers, the canned beers contained
439
as a consequence of the scalping characteristics significantly higher amounts of particularly
440
myrcene at a confidence level of 95 %, also because this was the only hop mono terpene
441
tested which arose at higher levels. It is noteworthy to mention that a 0.33 L beer can
442
possesses a total coating surface area of ca. 31.26 cm2 while a crown cork lining has a surface 21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
443
area of ca. 2.25 cm2. When taking the surface to volume ratios into consideration, it can be
444
stated that the total amounts of hop volatiles scalped per cm2 are considerably lower in the
445
beer can than in the crown cork liner. It should also be noted that the sum of all individual hop
446
volatiles in both the packaging (can coating or crown cork liners) and the beers (bottled or
447
canned beer) revealed significant differences at a 95 % confidence level. Myrcene, limonene,
448
linalool, α-terpineol, and α-humulene were higher in the bottled beer plus crown cork liner
449
polymers while the amount of t-caryophyllene was higher in the canned beer plus can
450
coatings. Geraniol formed an exception and no significant difference at the 95 % confidence
451
level was found (Table 4).
452
The possibility that analytes are removed by the extensive washing step of the cans was tested
453
in a separate trial. The washing step was therefore done according to the normal procedure to
454
rinse-off all adhesive analytes. Subsequently, the rinsed can material was washed with water
455
thoroughly again and the water from the second washing-step was extracted using
456
diethylether and analyzed. None of the analytes were identified in the concentrated extracts at
457
detectable levels. The washing-step is consequently most likely not responsible for the lower
458
‘total’ amounts of most analytes in the canned beer (plus coating) than in the bottled beers
459
(plus liner). A reasonable explanation for this observation can therefore not be given.
460 461
The series of experiments presented clearly demonstrated that hop volatiles are prone to
462
migration into beer packaging but to a deviant degree. In all closures tested within the scope
463
of this study, scalping of constituents occurred to a high extent, and differences between the
464
closures were only little relative to the degree of losses. The shelf-life of beer is correlated
465
with the preservation of its original aroma. Scalping causes the loss of certain volatiles thus
466
provoking a disturbance of the original balance between the aroma compounds. Linalool and
467
geraniol may have a key role for hop aroma perception in beer21 and were hardly lost to the 22 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
468
packaging, but particularly in aromatic beers, many aroma-active constituents make up the
469
characteristic flavor and aroma in a complex interplay. It is very likely that in addition to the
470
compounds monitored in this investigation, also other beer constituents are being scalped and
471
lost to the packaging as e.g. reported in.11 The impacts of this study’s outcomes on aroma
472
alterations are therefore undeniable. Deductions from this thus relate to the role of flavor
473
scalping and point to the importance to improve closures to enhance aroma consistency and
474
stability of packaged beer. Also, there is a need for awareness when assessing aroma in food
475
systems as key aroma compounds may be depleted before being analyzed.
476 477
ABBREVATIONS USED
478
HDPE, high-density polyethylene; LDPE, low-density polyethylene; PE, polyethylene; PP,
479
polypropylene; PVC, poly vinyl chloride.
480 481
ACKNOWLEDGEMENT
482
The authors wish to thank Pelliconi & C. S.p.A. for providing crown corks for this study.
483
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
484
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during high pressure treatments. J. Food Eng. 2011, 102, 9-15.
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46.
613 614
polyethylene. Food Addit. Contam. 2006, 23, 738-746. 47.
615 616
Limm, W.; Begley, T. H.; Lickly, T.; Hentges, S. G., Diffusion of limonene in
Van Willige, R. W. G. Effects of flavour absorption on foods and their packaging materials. Dissertation, Wagening University, Wagening, The Netherlands, 2002.
48.
Lam, K. C.; Deinzer, M. L., Tentative identification of humulene diepoxides by
617
capillary gas chromatography/chemical ionization mass spectrometry. J. Agric. Food.
618
Chem. 1987, 35, 57-59.
619
49.
620 621 622
Kaneda, H.; Kobayashi, N.; Takashio, M.; Tamaki, T.; Shinotsuka, K., Beer staling mechanism. Tech. Q. Master Brew. Assoc. Am. 1999, 36, 41-47.
50.
Narziß, L.; Miedaner, H.; Eichhorn, P., Untersuchungen zur Geschmacksstabilität des Bieres (Teil 2). Mschr. Brauwiss. 1999, 5/6, 80-85.
29 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
623
Figure captions
624 625
Figure 1: Hop monoterpene, sesquiterpene and terpene alcohol concentrations absorbed into
626
an LDPE liner polymer and concentrations in beer as a function of storage time; a: myrcene;
627
b: limonene; c: linalool; d: α-terpineol; e: geraniol; f: t-caryophyllene; g: α-humulene. ()
628
amounts in liner, experimental data; () fit of the Fickian model to the amounts found in the
629
liner. () amounts in beer, experimental data. Data points with asterisks were below the limit
630
of quantification. Mean values are presented and error bars represent ± 1 standard deviation, n
631
= 3.
632 633
Figure 2: Comparison of the accumulation of hop volatiles in four different crown cork liner
634
materials after 55 days of storage. White bars: LDPE-liner from manufacturer A; hatched
635
bars: LDPE-liner from manufacturer B; light grey bars: LDPE-oxygen scavenging liner; dark
636
grey bars: HDPE-oxygen barrier liner. Results from linalool, α-terpineol, and geraniol are
637
magnified to ease visibility of the bars. Bars with asterisks were below the limit of
638
quantification. Mean values are presented and error bars represent ± 1 standard deviation,
639
letters above bars indicate statistical significance at p < 0.05, n = 3.
640 641
Figure 3: Influence of four different crown cork liner materials on the composition of hop
642
volatiles in beer after 55 days of storage. White bars, LDPE-liner from manufacturer A;
643
hatched bars, LDPE-liner from manufacturer B; light grey bars, LDPE-oxygen scavenging
644
liner; dark grey bars, HDPE-oxygen barrier liner. Mean values are presented and error bars
645
represent ± 1 standard deviation, letters above bars indicate statistical significance at p < 0.05,
646
n = 3.
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Journal of Agricultural and Food Chemistry
Tables
Table 1: Retention times and m/z ratios used for analyzing hop volatiles by SAFE-GC/MS. Substance
tR [min]
m/z
myrcene
23.87
93
4-octanol
23.96
73
limonene
24.92
68
linalool
26.27
93
α-terpineol
28.16
59
geraniol
29.44
69
t-caryophyllene
34.18
93
α-humulene
35.05
93
tR = retention time.
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Journal of Agricultural and Food Chemistry
Page 32 of 38
Table 2: Hop volatiles in liner polymer after 24 and 72 hours incubation at room temperature. The incubation for 24 hours was done in a duplicate trial and values are given as the mean. The incubation for 72 hours was done as a single trial. incubation for 24 hours
incubation for 72 hours
liner [µg]
% in liner
liner [µg]
% in linera
LDPE-myrcene
8.4
6.5
11.3
8.7
LDPE-limonene
9.1
7.0
13.4
10.3
LDPE-linalool
1.8
1.4
2.1
1.6
LDPE-geraniol
1.5
1.2
1.6
1.2
LDPE-α-terpineol
2.5
1.9
2.7
2.1
LDPE-t-caryophyllene
6.2
4.8
10.5
8.1
compound
a
LDPE-α-humulene 7.2 5.5 13.0 10.0 a : percentages were calculated based on the absolute amount in the solutions of 130 µg.
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Journal of Agricultural and Food Chemistry
Table 3: Model parameters as deduced Fick’s second law (eq. 1). The D-values of tcaryophyllene and α-humulene were derived from non-linear fitting of eq. 1 to the experimental data. All values shown are calculated.
Polymer-volatile
D ± Std. Dev. [cm2/day]
Mmax ± Std. Dev. [µg]
t0.99 [days]
RMSE/Mmax ×100 [1/µg]
LDPE-myrcene
1.17 e-5 ± 0.19 e-5 a
144.0 ± 18.5 a
21.6 ± 3.9
13.3
LDPE-limonene
1.32 e-5 ± 0.16 e-5 a
275.8 ± 29.3 b
LDPE-linalool
-5
-5 b
-5
-5 bc
0.84 e ± 0.07 e
LDPE-α-terpineol
0.72 e ± 0.07 e
LDPE-geraniol
0.49 e-5 ± 0.09 e-5 cd -5
-5 d, 1
-5
-5 d, 1
LDPE-t-caryophyllene 0.32 e ± 0.11 e
18.7 ± 2.4
19.1
3.9 ± 0.3
c
29.6 ± 2.4
3.1
1.6 ± 0.1
c
34.3 ± 3.3
2.3
50.5 ± 9.1
6.0
78.0 ± 27.1
9.7
1.1 ± 0.0 c 184.4 ± 3.7
d e
LDPE-α-humulene 0.26 e ± 0.06 e 227.0 ± 5.9 96.2 ± 24.8 9.7 : diffusion coefficients were determined using non-linear fitting. D = diffusion coefficient;
1
Mmax = amount of volatile in the liner material at equilibrium; RMSE = root mean square deviation of the modeled data from the experimental data; t0.99 = time at which 99 % of Mmax was reached. Mean values ± 1 standard deviation are present and letters in apex indicate statistical significance at p < 0.05, n = 3.
33 ACS Paragon Plus Environment
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Table 4: Amount of hop volatiles found and percent absorption in bottled beer, crown cork liner materials, canned beer, and can coatings after 46 days of storage. bottled beer beer [µg]
crown liner [µg]
canned beer
% in beer a
myrcene
8.9 ± 1.0
279.4 ± 34.8
3.1 ± 0.3
limonene
0.3 ± 0.0
2.3 ± 0.2
12.1 ± 1.1 a
200.9 ± 8.9
1.2 ± 0.2
α-terpineol
6.0 ± 0.1
geraniol
% in liner
can coating [µg]
% in beer
% in coating b
71.2 ± 9.1 b
41.0 ± 1.1
101.6 ± 13.0
28.8 ± 0.8
87.9 ± 6.6 a
0.5 ± 0.0
0.6 ± 0.0
46.3 ± 4.2 b
53.7 ± 2.7 b
99.4 ± 4.4 a
0.6 ± 0.1 a
169.4 ± 7.7
0.7 ± 0.1
99.6 ± 4.5 a
0.4 ± 0.1 a
< 0.3
100 ± 1.6 a
n.d. a
5.6 ± 0.4
< 0.3
100 ± 6.4 a
n.d.
71.5 ± 4.5
0.8 ± 0.2
98.9 ± 6.3 a
1.1 ± 0.2 a
69.2 ± 6.7
0.5 ± 0.4
99.3 ± 9.7 a
0.7 ± 0.6 a
t-caryophyllene
< 0.3
1.3 ± 0.1
n.d.
100.0 ± 7.8 a
< 0.3
8.7 ± 0.2
n.d.
100.0 ± 2.7 a
α-humulene
< 0.3
5.3 ± 0.1
n.d.
100.0 ± 2.3 a
0.4 ± 0.0
0.8 ± 0.1
35.0 ± 2.0 b
65.0 ± 8.1 b
linalool
96.9 ± 12.1
beer [µg] a
n.d. = not detected. Mean values ± 1 standard deviation are presented. Letters in apex indicate significant difference of mean percentages between canned beers and bottled beers, and can coatings and crown cork closures, respectively, by a Student’s t-test at the 95 % confidence level, n = 3.
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Journal of Agricultural and Food Chemistry
Figures
0
75
50
0
60
10
20
30
40
50
150
1 0
0
0
storage [days] 1.2
2
125 0
60
10
40
50
190
0.9
180 0.6 170
*
160
0.0
150 10
20
30
40
storage [days]
50
60
f
200
60
150
45
100
30
50
15
0
0 0
10
200 0.5
175
*
0.0
60
150 0
10
20
30
40
50
60
storage [days]
Figure 1
35
ACS Paragon Plus Environment
20
30
40
storage [days]
200
e
0
30
225 1.0
storage [days]
storage [days]
0.3
20
α-humulene in liner [µg]
40
175
t-caryophyllene in beer [µg]
30
t-caryophyllene in liner [µg]
20
15
geraniol in beer [µg]
10
30
3
250
1.5
g
250
60
200 45 150 30
100
15
50 0
0 0
10
20
30
40
storage [days]
50
60
50
60
α-terpineol in beer [µg]
150
0 0
45
200
275
d
α-humulene in liner [µg]
10
225
4
2.0
α-terpineol in liner [µg]
50
60
225
c
linalool in beer [µg]
20
300
5
75
b
linalool in liner [µg]
100
limonene im liner [µg]
30
myrcene in beer [µg]
150
375
limonene in beer [µg]
40
a
geraniol in liner [µg]
myrcene in liner [µg]
200
Journal of Agricultural and Food Chemistry
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400
b
amount in liner [µg]
a
a
300
a
a
a
b
200
a
a a
a a
a
4
a
3
a a
a
b b
2
100
c
1
a ab
b c
0
a b* bc * * c
linalool α-terpineol geraniol
0 myrcene
limonene
linalool
α-terpineol
geraniol
Figure 2
36 ACS Paragon Plus Environment
t-caryophyllene
α-humulene
Page 37 of 38
Journal of Agricultural and Food Chemistry
300
a a
amount in beer [µg]
250
a 200
a a
a a a a a a a
150
100
50
a a a a
a a a a
a a a a
a a a a
t-caryophyllene
α-humulene
0 myrcene
limonene
linalool
α-terpineol
geraniol
Figure 3
37 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
TOC Graphic
38 ACS Paragon Plus Environment
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