The Influence of Hopping on Formation of ... - Wiley Online Library

The Influence of Hopping on Formation of Carbonyl Compounds During Storage of Beer Alexandr Mikyška1,*, Karel Krofta2, Danuša Hašková1, Jiří Čulík1 and Pavel Čejka1 ABSTRACT

J. Inst. Brew. 117(1), 47–54, 2011 Improving beer flavour stability is an important brewing goal. Pilot scale brewing trials (50 L) were performed that focused on the determination of the influence of hop pellet dosage and dosage timing on carbonyl compounds in stored beer. The reducing activity of experimental worts, beers and stored beers appeared to depend on the hop pellet dose. Brews with lower amounts of hop antioxidants showed an enhanced formation of carbonyl compounds over the course of beer storage. A correlation between DPPH reducing activity and the content of some carbonyls, including the important markers 2-furfural and (E)-2-nonenal, was found. Fresh and aged beers hopped by different amounts of hop pellet doses were clearly distinguishable according to their carbonyl content using Cluster analysis. Results of the sensorial analysis corresponded to the analytical criteria values. Results of this study bring further evidence of the indispensable impact of hop antioxidants on the suppression of undesirable carbonyl compound formation in the course of beer staling, which can be significant in beers hopped by aroma hops. However, hop antioxidants are only one of many factors affecting beer staling. Key words: Antioxidants, beer ageing, carbonyls, flavour stability, free phenolic compounds, hopping, polyphenols.

phenols and phenolic substances29. Hop alpha bitter acids also exhibit weak antioxidant activity22,24. Polyphenol substances of malt7,16,25,27,28,32 and hops13,22,26,27 affect antioxidant activity and thus, potentially, sensorial stability of beer. Literature-based data suggest that approximately 70 to 80% of beer polyphenols have their origin in malt, 20 to 30% are hop polyphenols12, and that the proportion of hop polyphenols is probably higher26 if aroma hops rich in polyphenols have been used. Some authors conclude that deceleration of undesirable processes in the course of beer storage is mostly affected by sulphur dioxide, which is formed in the course of fermentation and that the significance of polyphenolic and sugar antioxidants originating from raw materials is considerably lower3,5,14,19,31. Other authors have found a clear relationship between antioxidative properties of intermediates of beer production and formation of stale flavour carbonyls and the sensorial stability of beer4,13,16,22,23,26,27,40,41. The aim of this study was to obtain more information on the impact of hop polyphenols and phenolic compounds on antioxidant activity and carbonyl formation in fresh and stored beer.

MATERIALS AND METHODS INTRODUCTION Improving beer flavour stability is an important task of brewing researchers. Free radical reactions have been proposed as the main cause of beer stale flavour production17. Carbonyl compounds are formed in radical chain reactions by active oxygen species action on substances such as fatty acids, amino acids, higher alcohols and saccharides39. Polyphenols and phenolic compounds are reputed to play an important role in flavour stability because of antioxidant activity. The role of composition of antioxidants and antioxidative properties of brewing raw materials in flavour deterioration is only partially known. Polyphenol substances, sugar reductons and the sulphur dioxide formed during fermentation are important natural antioxidants in brewing. The main antioxidants in hops are poly-

Brewing trials A series of five experimental 50 L brews of 12°P all malt pale lager beers with different hopping options were prepared (Table I). Brews were hopped by the use of 100% CO2 hop extract (Magnum) and/or 50% and 100% hop pellets (Saaz). Pellet application, either in one dose at the beginning of wort boiling or a dose divided into three portions, was compared to obtain information about the hop polyphenol role in the course of wort boiling from the point of view of the antioxidant action. Considering the large losses of iso-humulones in our experimental brewery and the average bitterness of Czech commercial lager Table I. Overview of hopping regimes for experimental beers. Code

1

Research Institute of Brewing and Malting, Lípová 15, 120 44 Praha 2, Czech Republic. 2 Hop Research Institute, Kadaňská 2525, 438 46 Žatec, Czech Republic. * Corresponding author. E-mail: [email protected] Publication no. G-2011-0314-1050 © 2011 The Institute of Brewing & Distilling

EX PE-EX-1 PE-EX-2 PE-1 PE-2

Hop product

Amount

Timing

CO2-extract (Magnum) CO2-extract (Magnum) Pellets 90 (Saaz) CO2-extract (Magnum) Pellets 90 (Saaz) Pellets 90 (Saaz) Pellets 90 (Saaz) Pellets 90 (Saaz) Pellets 90 (Saaz) Pellets 90 (Saaz)

100% 50% 50% 50% 25% 25% 100% 50% 25% 25%

Start Start Start Start 30 min after start 20 min before end Start Start 30 min after start 20 min before end

VOL. 117, NO. 1, 2011

47

beers at 26 BU10, a compromise hop raw material dose was calculated on 10 mg of alpha-acids per 1 L. The resulting bitterness of the final beers was 22 ± 2 EBC units. Wort boiling took 90 min. Break was separated in a clarifying tank; cooled wort was aerated to the same level of dissolved oxygen (7 mg/L) and inoculated by pressed yeast, strain No 95 of the RIBM brewing yeast collection. The sensorial profile of the beer is formed primarily in the course of fermentation and maturation. Formation or decomposition of sensorial active substances depends both on their content and on content of precursors in wort, as well as on the physiological stage and the yeast strain, the yeast pitching dose and the fermentation temperature course. Detectability of the chemical composition effect on beer sensorial properties decreased from beer to raw materials used for production. Influence of different physiological stages of yeast was reduced to a minimum. For all brews, fresh pressed yeast after the 1st pitching was used. Maturation was 40 days at a temperature 0–2°C. Beers were filtered and bottled by the use of a filling machine with double-evacuation and pre-filling of bottles by carbon dioxide. Bottled beer was pasteurized at ~20 PU. Beers were stored in the laboratory at a temperature of 20°C. Analyses Analyses of hop products, hopped worts and beers including total polyphenols and flavanoids were made according to Analytica EBC1, anthocyanogens were determined according to Pivovarsko-Sladařská Analytika6. Polyphenols, free phenolics and antioxidant power of hop materials were analyzed in boiling water extracts (5 g/L, 30 min boil under a reflux cooler)29. Content of free phenolic substances (gallic acid, protocatechuic acid, gentisic acid, esculin, p-hydroxybenzoic acid, 4-hydroxyphenylacetic acid catechin, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, epicatechin, vanillin, coumaric acid, umbelliferon, scopoletin, ferulic acid, sinapic acid, rutin, naringin, myricetin, 4-hydroxycoumarin, daidzein, quercetin, genistein, apigenin, formononethin, biochanin A) were determined by HPLC with a coulometric detector20. Reducing (antiradical) activities of hop raw materials, worts and fresh and stored beers were measured by three different methods, each of them determined a slightly different spectrum of antioxidant or antiradical power. Reducing capacity (2,6-dichlorphenol indophenol, DCPI) was measured according to Analytica MEBAK8. Reducing activity was measured by the use of stable free radical DPPH (1,1-dipyridyl-2-picryl hydrazyl) according to the ESR-DPPH procedure previously described29,30. Determination of endogenous antiradical capacity of worts (T150 value) and beers (lag-time value) proceeded according to the original method described by Ushida37,38. Sugar reductons and melanoidins were determined by the DCPI method16,25. The DPPH reducing activity value concerns mainly slow reducing substances, primarily polyphenols17,30. In the determination of endogenous antiradical capacity of the beers, the lag-time value primarily represents the influence of SO2 and oxygen charge3,5. Sensorial active higher alcohols and esters contained in beer were determined by the GC method2. Thirteen car48

JOURNAL OF THE INSTITUTE OF BREWING

bonyl compound markers, (2-methylpropanal, 2-methylbutanal, 3-methylbutanal, benzaldehyde, phenylacetaldehyde, (E)-2-nonenal, (E)-2-octenal, (E)-butenal, hexanal, heptanal, octanal, 2-furfural, 3-methyl butan-2-on) were determined by a GC/MS method11,35. Sensorial analysis of fresh beer and beer after 3 and 6 months storage was carried out by a panel of eight trained RIBM tasters. Panellists were instructed to focus their evaluation on off-flavours and on the overall acceptance of the sensorial profile of the beers and at the same time to eliminate differences in hop flavour and bitter taste intensity. With respect to the need for simultaneous evaluation of whole sets of carbonyl compounds, multivariate data analysis methods and cluster analysis were applied.

RESULTS AND DISCUSSION Two hop raw materials, extreme in polyphenol content, were chosen for hopping. The Saaz hop variety is distinguished by its high content of polyphenols and reducing activity value21, as can be seen from the results of the hop analysis in Table II. Hop CO2 extract showed negligible values for polyphenol compounds and reducing power. A wide range of free phenolic compounds from a group of flavonoids and phenolic acids was monitored. Some determined free phenolic compounds which could be potential antioxidants and are as follows: flavan-3-ols (catechin, epicatechin), flavonols (quercetin, myricetin, rutin), flavanones (naringin), isoflavones (daidzein, genistein, formononethin, biochanin A), hydroxycinnamic acids (ferulic, coumaric, caffeic, sinapic acids) and p-hydroxybenzoic acids (gallic, protocatechuic, gentisic, syringic, vanillic acids). Although results of experiments dealing with the antioxidant action of free phenolic compounds have been published, knowledge in this area is not sufficient. Results of hop materials and sweet wort analyses demonstrate differences in the free phenolic compound composition between hops and malt, as well as hop pellets and extract (Table III). A main part of the free phenolics in hop extract is the flavonoids (85%), namely flavonols (28%) and isoflavonoids (40%), but the total content of these compounds was very low (0.2 mg/g). Saaz pellets were high in flavanoids (70%), namely flavan-3-ols (45%) and the total amount of free phenolics was 3.5 mg/g. Free phenolic compounds in sweet wort have comparable amounts of flavonoids (47%) and phenolic acids, hydroxycinnamic acids (23%) and hydroxybenzoic acids (20%). The content of total polyphenols, anthocyanogens (Table IV) and all groups of free phenolic compounds (Table

Table II. Analyses of hop products. Hop productsa Alpha-acids (%) Beta-acids (%) Total polyphenols (mg/L) Anthocyanogens (mg/L) RCDCPI (% rel.) ESR RADPPH (% rel.) ESR-T150 a Polyphenols

Sweet wort

CO2-extract

Pellets 90

11.7°P

50.3 28.0 7.2 8.5 3 2 0.10

3.9 4.7 276 123 19 83 1.98

174 64 51 61 18.72

and antioxidant activity of boiling water extract 5 g/L.

V) in hopped worts depended on the dose of hop pellets and the amount of polyphenols in the hopping. Anthocyanogen content was proportional to hop pellet dosage. Total polyphenol concentration showed a non-linear dependence. Polyphenol precipitation degree increased with the applied pellet dosage. Both assumed content of free phenolic compound markers and the flavanoid content increase was prominent. A double amount of total free phenolic compounds (48 mg/L) was detected in wort hopped wholly by hop pellets (PE-1, PE-2 options) in comparison with CO2 hop extract option (EX). Differences between malt and hop free phenolic composition resulted in different ratios of flavonoids and phenolic acids in hopped worts and the share of flavonoids increased with the pellet dose. Clear differences were not found

between polyphenols, or as the free phenolic compound content in the worts hopped either by a single dose or by a divided dose. Only a weak trend to lower anthocyanogen content was observed in beers hopped by the divided dose. Thus the effect of a 25% pellet dosage, 20 min before end of boiling, and a dosage of 100% pellets at the beginning, had a similar effect from the point of view of polyphenol release from the hop pellets and their precipitation in the course of wort boiling and fermentation. Prepared non-stabilized beers had a natural colloidal stability from 4.5 till 6.5 months. A negative impact of hop polyphenols was apparent in the samples with a full pellet dose (Table VI). Bitterness of beers prepared by the divided hop pellet dose (PE-EX-2, PE-2) was slightly lower in BU compared

Table III. Concentration of free phenolic compounds in hop products and sweet wort. Hop productsa CO2-Extract Catechin Epicatechin Rutin Myricetin Quercetin Naringin Apigenin Daidzein Genistein Formononethin Biochanin A Ferulic acid Sinapic acid Coumaric acid Chlorogenic acid Caffeic acid p-Hydroxybenzoic acid Gallic acid Protocatechuic acid Gentisic acid Vanillic acid Syringic acid 4-Hydroxycoumarin Umbelliferon Esculin Scopoletin Vanillin 4-Hydroxyphenylacetic acid Total a

Sweet wort Pellets 90

11.7°P

mg/L

%

mg/L

%

mg/L

%

0.00 0.00 0.17 0.00 0.11 0.12 0.05 0.06 0.03 0.20 0.11 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.05 0.05 0.00 1.00

0.0 0.0 17.0 0.0 11.0 12.0 5.0 6.0 3.0 20.0 11.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 5.0 5.0 0.0 100.0

6.05 1.81 1.17 0.00 0.11 1.05 0.55 0.06 0.03 1.21 0.11 0.21 0.05 0.40 0.39 0.11 0.00 0.00 0.04 0.04 0.47 0.72 0.00 0.13 0.80 0.15 0.59 1.11 17.36

34.9 10.4 6.7 0.0 0.6 6.0 3.2 0.3 0.2 7.0 0.6 1.2 0.3 2.3 2.2 0.6 0.0 0.0 0.2 0.2 2.7 4.1 0.0 0.7 4.6 0.9 3.4 6.4 100.0

4.30 0.40 2.60 0.00 0.10 1.20 0.00 0.00 0.00 0.00 0.00 3.40 0.60 0.00 0.00 0.20 2.90 0.00 0.10 0.00 0.50 0.10 1.00 0.10 0.20 0.10 0.10 0.50 18.40

23.4 2.2 14.1 0.0 0.5 6.5 0.0 0.0 0.0 0.0 0.0 18.5 3.3 0.0 0.0 1.1 15.8 0.0 0.5 0.0 2.7 0.5 5.4 0.5 1.1 0.5 0.5 2.7 100.0

Boiling water extract 5 g/L.

Table IV. Hopped wort analyses. Extract (% wt) pH Bitterness (BU) Total polyphenols (mg/L) Anthocyanogens (mg/L) RCDCPI (% rel.) ESR RADPPH (% rel.) ESR-T150

EXa

PE-EX-1b

PE-EX-2c

PE-1d

PE-2e

12.38 5.78 57 180 58.3 54 65 9.16

12.35 5.82 54 298 82.1 57 77 4.75

12.59 5.60 51 283 78.7 60 78 3.62

12.73 5.77 49 410 95.8 61 83 7.25

12.66 5.77 51 434 118.8 60 85 5.35

a CO extract. 2 b Pellets + CO

2 extract, single dose. + CO2 extract, divided dose. d Pellets, single dose. e Pellets, divided dose. c Pellets

VOL. 117, NO. 1, 2011

49

Table V. Concentration of free phenolic compounds in hopped worts. EXa Catechin Epicatechin Rutin Myricetin Quercetin Naringin Apigenin Daidzein Genistein Formononethin Biochanin A Ferulic acid Sinapic acid Coumaric acid Chlorogenic acid Caffeic acid p-Hydroxybenzoic acid Gallic acid Protocatechuic acid Gentisic acid Vanillic acid Syringic acid 4-Hydroxycoumarin Umbelliferon Esculin Scopoletin Vanillin 4-Hydroxyphenylacetic acid Total

PE-EX-1b

PE-EX-2c

PE-1d

PE-2e

mg/L

%

mg/L

%

mg/L

%

mg/L

%

mg/L

%

4.1 0.9 2.6 0.0 0.1 1.3 0.0 0.0 0.0 0.0 0.0 3.4 0.6 0.1 0.0 0.2 2.2 0.3 0.2 0.0 0.8 0.1 1.5 0.5 0.3 0.0 0.6 0.5 20.2

20.2 4.6 12.8 0.0 0.7 6.2 0.0 0.0 0.0 0.0 0.0 16.7 2.8 0.5 0.0 0.9 11.1 1.3 0.8 0.1 3.8 0.7 7.4 2.4 1.4 0.0 3.0 2.6 100

7.8 2.1 2.4 0.0 0.0 6.2 0.3 0.0 0.1 0.0 0.0 3.0 0.6 0.9 0.3 0.2 0.0 0.3 0.2 0.0 0.6 0.1 0.9 0.2 0.1 0.3 0.1 0.5 27.1

28.7 7.8 8.9 0.0 0.0 23.0 1.2 0.0 0.5 0.0 0.0 11.3 2.0 3.5 1.3 0.6 0.0 1.0 0.8 0.1 2.1 0.3 3.3 0.7 0.3 1.1 0.4 1.7 100

8.5 2.0 2.6 0.0 0.1 5.1 0.3 0.0 0.2 0.3 0.0 3.2 0.6 0.4 0.2 0.2 0.0 0.3 0.2 0.0 0.8 0.1 2.1 0.2 0.1 0.4 0.2 0.5 28.6

29.8 6.9 9.0 0.0 0.5 17.8 1.1 0.0 0.6 0.9 0.2 11.1 2.1 1.3 0.8 0.6 0.0 0.9 0.8 0.1 2.9 0.5 7.3 0.7 0.4 1.3 0.6 1.7 100

12.5 3.3 1.4 0.0 0.0 16.6 0.4 0.1 0.1 4.3 0.0 3.3 0.6 1.0 0.8 0.2 0.0 0.3 0.3 0.0 0.7 0.1 0.2 0.3 0.2 0.0 0.7 0.5 48.0

26.0 7.0 3.0 0.0 0.0 34.6 0.8 0.2 0.3 9.0 0.0 6.8 1.3 2.1 1.7 0.4 0.0 0.6 0.6 0.0 1.5 0.1 0.5 0.7 0.3 0.0 1.4 1.1 100

12.3 3.1 2.7 0.0 0.1 19.6 0.3 0.0 0.2 0.5 0.0 3.2 0.6 1.0 0.6 0.2 0.0 0.3 0.3 0.0 0.7 0.1 0.5 0.3 0.1 0.3 0.2 0.5 47.6

25.9 6.4 5.6 0.0 0.3 41.1 0.6 0.0 0.4 1.0 0.0 6.7 1.3 2.1 1.3 0.4 0.0 0.6 0.6 0.0 1.4 0.2 1.0 0.5 0.2 0.7 0.5 1.1 100



Table VI. Results of experimental beers analyses. Original extract (% wt) Apparent extract (%) Real extract (%) Alcohol (% wt) Alcohol (% v) Apparent attenuation (%) Real attenuation (%) Colour (EBC) pH Total polyphenols (mg/L) Anthocyanogens (mg/L) Carbon dioxide (%) Sulphur dioxide (mg/L) RCDCPI (% rel.) ESR RADPPH (% rel.) ESR lag-time (min.) Higher alcohols (mg/L) Esters (mg/L) A/E ratio Shelf life (days)

EXa

PE-EX-1b

PE-EX-2c

PE-1d

PE-2e

12.30 2.70 4.50 4.01 5.12 78.1 64.6 7.6 4.66 155 42.4 0.50 6.5 53 54 38 82 20 4.0 185

12.25 2.56 4.43 4.04 5.16 79.1 65.3 7.4 4.67 228 66.1 0.51 7.4 59 65 42 84 24 3.5 165

12.46 2.91 4.75 3.99 5.10 76.7 63.4 7.5 4.65 229 64.4 0.50 7.0 64 66 32 84 26 3.2 195

12.62 2.74 4.65 4.13 5.28 78.3 64.8 8.3 4.69 310 76.5 0.50 6.2 65 74 35 86 22 3.8 140

12.07 2.54 4.37 3.97 5.07 79.0 65.2 7.7 4.75 290 71.7 0.48 6.9 58 73 40 79 22 3.6 135



with the corresponding one dose option. In the course of 6 months storage, the decrease of bitterness (1–2 BU) in all of the trial beers was similar (Table VII). The reducing capacity DCPI values of hopped worts, as well as fresh beers, did not show any dependence on 50


the hopping variation used, while the DPPH reducing activity value increased with the hop pellet dose (Table IV, VI, Fig. 1, 2). The increase in beers of PE-EX and PE options was 20% and 37% respectively. Differences between single dose and divided dose options were negligi-

Table VII. Changes in bitterness (BU) of experimental beers in the course of storage. Fresh beer 3 months storage 6 months storage

EXa

PE-EX-1b

PE-EX-2c

PE-1d

PE-2e

25 24 24

23 22 21

20 19 18

23 22 22

21 21 20



Fig. 1. Changes in reducing capacity DCPI from hopped worts to 6 month stored beers. Legend: CO2 Extract (▲); Pellets + CO2 Extract, single dose (■); Pellets + CO2 Extract, divided dose (□); Pellets, single dose (●); Pellets, divided dose (○).

Fig. 2. Changes in reducing activity ESR-DPPH from hopped worts to 6 month stored beers. Legend: CO2 Extract (▲); Pellets + CO2 Extract, single dose (■); Pellets + CO2 Extract, divided dose (□); Pellets, single dose (●); Pellets, divided dose (○).

ble. The antiradical capacity, T150 value of hopped worts, was affected by the hopping technology, with both the pellet application and time of dosage having some impact. The lowest (best) values were found in the PE-EX combinations, with the highest in the EX option. The ESR-T150 values of the worts hopped by the divided hop dose were slightly better than the ones hopped with one dose. The lag time values showed no dependence on the hopping schedule and the differences among the trial beers in lagtime values were up to 20%. (Table VI). The sulphur dioxide content in the fresh beers ranged from 6.2 to 7.4 mg/L.

A sudden decrease of DCPI reducing capacity value was detected in beers hopped by 100% and 50% hop extract, after a period of three months storage. The reducing capacity values decreased in beers hopped by 100% pellets in the course of six months storage (Fig. 1). DPPH reducing activity of worts and beers depended closely on the hop pellet dose and reducing activity decreased in the course of beer storage. Some differences were found in the decrease dynamics related to the hop pellet ratio and the value of the PE options decreased slowly (20% rel.) compared to the EX and PE-EX options (32%, 30% rel.). A trend related to hopping technology was not observed (Fig. 2). The higher alcohol and ester content was comparable in all fresh beers and there were not any relevant differences among the beer fermentations (Table VI). Carbonyl compounds occur in beer in different concentrations and they have diverse threshold levels of sensorial perception. It is possible to separate carbonyl compounds according to their precursors into several groups33,39. Precursors of Strecker aldehydes are amino acids and higher alcohols (CARB-AA: 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, benzaldehyde, phenylacetaldehyde), saturated fatty acids (CARB-FA1: hexanal, heptanal octanal), unsaturated fatty acids (CARB-FA2: (E)-2-nonenal, (E)-2-octenal, (E)-butenal,) and saccharides (2-furfural). Ketone 3-methyl-butan-2-on is considered to be a strong beer staling indicator by some authors33. Most of the carbonyl markers detected in the fresh and especially in the stored beer exhibited a decreasing trend in dependence on hop pellet usage. Comparison of all the above-mentioned carbonyl groups and individual markers in fresh and stored beers is given in Table VIII. Values of (E)-2 nonenal, often discussed in the literature, are included in CARB FA2 in addition to being given separately. An influence of hop pellet dose (PE versus PE-EX) on the CARB-AA group was observed after 6 month of storage. The amounts of the CARB-FA1 group did not show a clear dependence on hopping. The (E)-2 nonenal, as well as the CARB-FA2 group, evinced an unambiguous dependence on the hop pellet dose. In contrast, the 3methyl-butan-2-on content was lowest in the fresh beer hopped by hop extract and there was not any observed dependence of its content in stored beers on hopping variations. An evaluation of the raw material and brewing technology influence on trends in carbonyl formation was difficult, because individual carbonyl compound concentrations change during beer staling and depend on many reaction mechanisms, including temporary complexes with sulphur dioxide15,18,34,36. The reducing activity of the trial beers depended on the hop pellet dose. The content of some of the carbonyl compound markers correlated with the ESR-RADPPH reducing activity determined in fresh and stored beers (Table IX). There was an influence of hop pellet dosage timing on carbonyls in fresh beer (Fig. 3). Differences in the carbonyl content between beers hopped by different amounts of hop pellets (PE-EX versus PE options) were small compared with differences between the two tested variations in hop dosage timing (PE-1, PE-EX-1 versus PE-2, PE-EX-2). The PE-EX-2 variation was similar to the EX option. Reactions with hop antioxidants in the whole VOL. 117, NO. 1, 2011

51

Table VIII. Changes in carbonyl content (µg/L) of experimental beers stored for 3 and 6 months. EXa

PE-EX-1b

PE-EX-2c

PE-1d

PE-2e

23.0 55.2 105.7

19.0 36.9 98.5

20.6 37.2 97.7

20.2 40.4 88.3

21.4 37.4 82.7

19.2 19.0 24.8

22.6 22.3 25.1

17.0 20.2 24.2

17.4 13.2 30.6

16.5 14.2 24.1

14.6 23.6 17.5

12.9 18.0 16.4

12.7 16.7 16.3

10.0 16.8 12.1

10.9 15.3 13.7

4.5 4.2 8.3

3.2 3.9 7.6

3.2 3.9 7.6

1.8 3.5 5.6

1.6 3.1 5.9

5.3 48.0 84.6

4.1 38.0 59.5

3.7 37.0 73.2

4.5 36.0 42.0

4.1 34.0 40.6

0.7 0.2 2.7

0.9 0.2 1.9

1.0 0.2 2.4

1.5 0.7 2.8

1.7 0.4 2.2

CARB-AAf fresh beer 3 months 6 months CARB-FA1g fresh beer 3 months 6 months CARB-FA2h fresh beer 3 months 6 months (E)-2-Nonenal fresh beer 3 months 6 months 2-Furfural fresh beer 3 months 6 months 3-Methyl-butan-2on fresh beer 3 months 6 months a

CO2 extract. Pellets + CO2 extract, single dose. c Pellets + CO extract, divided dose. 2 d Pellets, single dose. e Pellets, divided dose. f 2-Methylpropanal, 2-methylbutanal, 3-methylbutanal, benzaldehyde, phenylacetaldehyde. g Hexanal, heptanal octanal. h (E)-2-Nonenal, (E)-2-octenal, (E)-butenal. b

Table IX. Dependence of carbonyl content in beer on ESR-RADPPH reducing activity. 2-Methyl-propanal 2-Methyl-butanal 3-Methyl-butanal Phenylacetaldehyde Benzaldehyde Hexanal Heptanal Octanal (E)-2-Butenal (E)-2-Octenal (E)-2-Nonenal 2-Furfural 3-Methyl-butan-2on CARB-AAc CARB-FA1d CARB-FA2e

Fresh

3 months

6 months

–0.22 –0.24 –0.15 –0.62 0 –0.48 0.71 0.18 –0.7 –0.93 –0.97 –0.63 0.87 –0.54 –0.42 –0.97

–0.67 –0.82a –0.84a –0.77a 0.14 –0.85 –0.23 –0.44 –0.91b –0.82a –0.88b –0.94b 0.73 –0.78a –0.6 –0.93b

–0.84a 0.64 –0.05 –0.80b 0.19 0.65 0.43 0.34 –0.91b –0.25 –0.97b –0.92b 0.13 –0.93b 0.56 –0.96b

at α = 0.05. b Significant at α = 0.01. c 2-Methylpropanal, 2-methylbutanal, 3-methylbutanal, benzaldehyde, phenylacetaldehyde. d Hexanal, heptanal, octanal. e (E)-2-Nonenal, (E)-2-octenal, (E)-butenal. a Significant

course of wort boiling are probably important for the carbonyl content level in the fresh beer. In the course of beer storage, carbonyl compound formation may depend more on the total hop pellet dose or pellet/extract ratio. The largest difference between a hop pellet dose of 100% and 50% was detected after three months of storage and relevant differences between the EX and PE-EX brews were found (Fig. 4). After six months of storage, the carbonyl content was similar to the 3 month stored beer, but the 52


Fig. 3. Differences in carbonyl compounds content of fresh beers (cluster analysis,13 markers). Legend: CO2 Extract (EX); Pellets + CO2 Extract, single dose (PE-EX-1); Pellets + CO2 Extract, divided dose (PE-EX-2); Pellets, single dose (PE-1); Pellets, divided dose (PE-2).

PE-EX option was closer to the EX option and the difference between the PE-1 and PE-2 option was higher. The lowest content of most of the carbonyl markers was in the beer hopped by a full pellet dose divided into three portions (Fig. 5, Table VIII). This is further evidence of the hop antioxidant impact on the suppression of carbonyl compound formation during beer staling. The relationship among total polyphenols, antiradical activity and carbonyls in beer is displayed in Fig. 6. Carbonyl content differences are shown in Figs. 3–5 and these corresponded to the sensorial evaluation of

Table X. Results of fresh and stored beer sensorial analysis (ranking test). Fresh EX PE-EX-1 PE-EX-2 PE-1 PE-2 Fcrit. = 9.49 (α = 0.05)

3 months

6 months

Rank sum

Rank

Rank sum

Rank

Rank sum

Rank

31 21 26 18 24 F = 4.9

5 2 4 1 3

29 25 19 24 23 F = 4.4

5 4 1 3 2

36 27 19 22 16 F = 12.3

5 4 2 3 1

Fig. 4. Differences in carbonyl compounds content of three month stored beers (cluster analysis, 13 markers). Legend: CO2 Extract (EX); Pellets + CO2 Extract, single dose (PE-EX-1); Pellets + CO2 Extract, divided dose (PE-EX-2); Pellets, single dose (PE-1); Pellets, divided dose (PE-2).

Fig. 6. Relative dependence between antiradical activity (%), total polyphenols (mg/L) and carbonyls (µg/L).

CONCLUSIONS

Fig. 5. Differences in carbonyl compounds content of six month stored beers (cluster analysis, 13 markers). Legend: CO2 Extract (EX); Pellets + CO2 Extract, single dose (PE-EX-1); Pellets + CO2 Extract, divided dose (PE-EX-2); Pellets, single dose (PE1); Pellets, divided dose (PE-2).

beers by a ranking test (Table X). Fresh beers prepared by single hop pellets dose were deemed to be slightly better when compared to beers prepared by a divided hop pellet dose. After three and six months of storage, a preference for the split dose, as well as preference for the full pellet dose, was apparent. Differences were significant only for extreme variations in the 6 month stored beers. In addition, some hop polyphenols oxidized during wort boiling can also have a negative impact on beer flavour staling9.

The reducing activity of experimental worts, beers and stored beers appeared to depend on the hop pellet dose. Brews with lower amounts of hop antioxidants showed an enhanced formation of carbonyl compounds over the course of beer storage. A correlation between DPPH reducing activity and the content of some carbonyls, including the important markers 2-furfural and (E)-2-nonenal, was found. Fresh and aged beers, hopped by different amounts of hop pellet doses, were clearly distinguishable according to their carbonyl content using Cluster analysis. Results of the sensorial analysis corresponded to the analytical criteria values. Results of this study bring further evidence of the indispensable impact of hop antioxidants on the suppression of undesirable carbonyl compound formation in the course of beer staling, which can be significant in beers hopped by aroma hops. Hop antioxidants are only one of many factors affecting beer staling. Fermentation conditions and the oxygen charge in brewing operations also have to be taken into consideration. ACKNOWLEDGEMENTS

This work was supported by The Czech Ministry of Agriculture, research project NAZV - 1B44061. REFERENCES 1. Analytica EBC, 5th edition, European Brewery Convention, Fachverlag Hans Carl: Nurnberg, 1998. 2. Analytica EBC, 5th edition, European Brewery Convention, Fachverlag Hans Carl: Nurnberg, 1998, Method 9.39.

VOL. 117, NO. 1, 2011

53

3. Andersen, M., Outtrop, H. and Skibsted, L., Potential antioxidants in beer assessed by spin trapping. J. Agric. Food Chem., 2000, 48, 3106-3111. 4. Araki, S., Kimura, T., Shimizu, Ch., Furusho, S., Takashio, M. and Shinotsuka, K., Estimation of antioxidative activity and its relationship to beer flavour stability. J. Am. Soc. Brew. Chem., 1999, 57, 34-37. 5. Back, W., Franz, O. and Nakamura, T., Das Antioxidative Potenzial von Beer. Brauwelt, 2001, 141(6-7), 209-215. 6. Basařová G., Pivovarsko Sladařská Analytika, Merkanta: Praha 1994. 7. Boivin, P., Malanda, M., Maillard, M. N., Berset, C., Richard, H., Hughes, M., Richard-Forget, F. and Nicolas, J., Role of natural antioxidants of malt in the organoleptic stability of beer. Proc. Eur. Brew. Conv. Congr., Brussels, IRL Press: Oxford, 1995, pp. 159-168. 8. Brautechnische Analysenmethoden, Band II, MEBAK: FreisingWeihenstephan, 1987, 96-97. 9. Callemien, D., Bennani, M., Counet, C. and Collin, S., Which polyphenols are involved in aged beer astringency? Assessment by HPLC and time-intensity method. Proc. Eur. Brew. Conv. Congr., Prague, Fachverlag Hans Carl: Nurnberg, CD-ROM, 2005, pp. 809-814. 10. Čejka, P., Kellner, V., Čulík, J., Horák, T. and Jurková, M., Characterizing a Czech-type beer. Kvasny prum., 2004, 50, 3-11. 11. Čulík, J., Jurková, M., Horák, T. and Kellner, V., Zkušenosti s využitím nových technik plynové chromatografie při analýze senzoricky aktivních látek. Část. II. Stanovení karbonylových sloučenin pomocí derivatizace nebo detekce detektorem GCECD a GC-MS. Využití HPLC při stanovení 2-furfuralu. Kvasny Prum., 1998, 44, 7-11. 12. De Keukeleire, D., Plant Polyphenols, Chemistry, Biology, Pharmacology. Academic Publishers: New York, 1999, pp. 739-760. 13. Dostálek, P. and Karabín, M., Impact of hop polyphenols and antioxidant properties of wort on formation carbonyl compounds during boiling process and storage of beer. Proc. Eur. Brew. Conv. Congr., Venice 2007, Fachverlag Hans Carl: Nurnberg, CD-ROM, 2007, pp. 923-930. 14. Franz, O. and Back, W., Stability index - a new approach to measure the flavour stability of beer. Tech. Q. Master Brew. Assoc. Am., 2003, 40, 20-24. 15. Hughes, P., Reducing power and sulphur compounds implication and function in beer. Cerevisia, 2000, 25, 59-66. 16. Kaneda, H., Kobayashi, N., Furusho, S., Sahara, H. and Koshino, S., Reducing activity and flavour stability of beer. Tech. Q. Master Brew. Assoc. Am., 1995, 32, 90-94. 17. Kaneda, H., Kobayashi, N., Takashio, M., Tamaki, T. and Shinotsuka, K., Beer staling mechanisms. Tech. Q. Master Brew. Assoc. Am., 1999, 36, 41-47. 18. Kaneda, H., Osawa, T., Kawakishi, S., Munekata, M. and Koshino, S., Contribution of carbonyl-bisulfite adducts to beer stability. J. Agric. Food Chem., 1994, 42, 2428-2432. 19. Kautovirta-Noira, A., Virtanen, H., Poyri, S., Lehtinen, P., Nurmi, T., Hartwaal, P., Reinikainen, P., Siirila, J. and Home, S., The increase of antioxidant activity during mashing - does it improve beer flavour stability? Proc. Eur. Brew. Conv. Congr., Prague, Fachverlag Hans Carl: Nurnberg, CD-ROM, 2005, pp. 709-720. 20. Kellner, V., Jurková, M., Čulík, J., Horák, T. and Čejka, P., Some phenolic compounds in Czech hops and beer of Pilsner type. Brewing Science, 2007, Jan/Feb, 5-10. 21. Krofta, K., Mikyška, A. and Hašková, D., Antioxidant characteristics of hops and hop products. J. Inst. Brew., 2008, 114, 160166. 22. Lermusieau, G., Liégeois, C. and Collin, S., Reducing power of different hop variants. Cerevisia, 2001, 26, 33-41. 23. Liégeois, C., Lermusieau, G. and Collin, S., Measuring antioxidant efficiency of wort, malt and hops against AAPH induced

54


24. 25. 26. 27. 28.

29. 30.

31.

32. 33. 34.

35. 36.

37. 38.

39. 40.

41.

oxidation of an aqueous dispersion of linoleic acid. J. Agric. Food Chem., 2000, 48, 1129-1134. Liu, Y., Gu, X., Tang, J. and Liu, K., Antioxidant activities of hops (Humulus lupulus) and their products. J. Am. Soc. Brew. Chem., 2007, 65, 116-121. Maillard, M. N. and Berset, C., Evolution of antioxidant activity during kilning: Role of insoluble bound phenolic acids of barley and malt. J. Agric. Food Chem., 1995, 43, 1789-1793. Mikyška, A., Hašková, D., Horák, T. and Jurková, M., Impact of hop raw material type on antioxidant behaviour of beer. Kvasny Prum., 2010, 56, 294-302. Mikyška, A., Hrabák, M., Hašková, D. and Šrogl, J., The role of malt and hop polyphenols in beer quality, flavour and haze stability. J. Inst. Brew., 2002, 108, 78-85. Mikyška, A., Hašková, D. and Prokeš, J., Antiradical properties of malt accessed by EPR methods. Proc. Eur. Brew. Conv. Congr., Prague, Fachverlag Hans Carl: Nürnberg, CD-ROM, 2005, pp. 764-773. Mikyška, A., Krofta, K. and Hašková, D., Evaluation of antioxidant properties of hop and hop products. Kvasny Prum., 2006, 52, 214-218 Mikyška, A., Krofta, K. and Hašková, D., Evaluation of antioxidant properties of raw hop and hop products. In: Acta Horticulturae (ISHS) 778. D. Havkin-Frenkel, Ed., Leuven International Society for Horticultural Science: Leuven, 2008, pp. 97-110. Nakamura, T., Franz, O. and Back, W., pH dependence of radical scavenging activity of polyphenols, phenolic acid and sulfite. Proc. Eur. Brew. Conv. Congr., Budapest, Fachverlag Hans Carl: Nurnberg, CD-ROM, 2001, pp. 612-620. Narziss, L., Reicheneder, E. and Lustig, S., Oxygen optimisation in wort preparation. Brauwelt Int., 1989, 3, 238, 240, 242, 244, 246-248, 250. Narziss, L., Miedaner, H, Eichhorn, P. and Lustig, S., Technologische Faktoren der Geschacksstabilitat. Mittel. Oster. Getr. Inst., 1997, 5/6, 52-64. Nyborg, M., Outtrup, H. and Dreyer, T., Investigations of the protective mechanism of sulfite against beer staling and formation of adducts with trans-2-Nonenal. J. Am. Soc. Brew. Chem., 1999, 57, 24-28. Ojala, M., Kotiaho, T., Siirila J. and Sihvonen, M-L., Analysis of aldehydes and ketones from beer as PFBOA derivatives. Talanta, 1994, 41, 1297-1309. Sieberle, P. and Komarek, D., Staling of beer aroma: A longknown, but still unresolved challenge in brewing science. Proc. Eur. Brew. Conv. Congr., Prague, Fachverlag Hans Carl: Nurnberg, CD-ROM, 2005, pp. 661-675. Ushida, M. and Ono, M, Improvement of oxidative flavour stability of beer - Role of OH-radicals in beer oxidation. J. Am. Soc. Brew. Chem., 1996, 54, 198-204. Ushida, M., Suga, S. and Ono, M., Improvement of oxidative flavour stability of beer - Rapid prediction method for beer flavour stability by electron spin resonance spectroscopy. J. Am. Soc. Brew. Chem., 1996, 54, 205-211. Wackerbauer, K. and Hardt, R., Radikalreaktionen und die Geschacksstabilitat des Bieres. Brauwelt, 1996, 136(40/41), 1880-1889. Walters, M. T., Heasman, A. P. and Hughes, P. S., Comparison of (+)-catechin and ferulic acid as natural antioxidants and their impact on beer flavour stability. Part 1: Forced-aging. J. Am. Soc. Brew. Chem., 1997, 55, 83-89. Walters, M. T., Heasman, A. P. and Hughes, P. S., Comparison of (+)-catechin and ferulic acid as natural antioxidants and their impact on beer flavour stability. Part 2: Extended storage trials. J. Am. Soc. Brew. Chem., 1997, 55, 91-98.

(Manuscript accepted for publication November 2010)