Changes in Key Aroma Compounds during Natural Beer Aging

Chapter 5

Changes in Key Aroma Compounds during Natural Beer Aging

Downloaded by EMORY UNIV on February 4, 2016 | http://pubs.acs.org Publication Date: October 10, 2002 | doi: 10.1021/bk-2003-0836.ch005

P. Schieberle and D. Komarek Deutsche Forschungsanstalt fuer Lebensmittelchemie, Lilchtenbergstrasse 4,85748 Garching, Germany

Application of Aroma Extract Dilution Analysis on a flavor extract isolated from a fresh pale lager beer revealed 41 odor -active compounds among which 2-phenylethanol (sweet, flowery), 4-hydroxy-2,5-dimethyl-2(5H)-furanone (caramel -like), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (seasoning-like) and (E)-ß-damascenone (cooked apple-like) predominated with the highest Flavor Dilution (FD)-factors. In a beer sample of the same batch stored for 34 month at 20°C in the dark, the concentrations of ten of the most odor-active compounds did not change significantly. However, four compounds, namely ethyl 2-methylpropanoate and ethyl 2-methylbutanoate with fruity notes as well as the Strecker aldehydes methional (cooked potato-like) and phenylacetaldehyde (sweet, honey -like) increased by factors of 3 to 10, respectively. Based on a very precise method developed for the quantitation of (E)-2nonenal, it could clearly be ruled out that this aldehyde contributes to off-flavor formation in the beer investigated.

Introduction Flavor staling of beer has been, and still is, one of the greatest challenges in the brewing industry. A great number of sensory studies have shown that during storage the beer flavor is shifted to more sweet, toffee-like notes, while the typical bitter, hoppy aroma is decreased. Furthermore, cardboard-like and ribeslike aromas may develop, in particular, when higher concentrations of oxygen are dissolved in beer (1-4). It is, however, obvious from most of the studies that the

70

© 2003 American Chemical Society

In Freshness and Shelf Life of Foods; Cadwallader, Keith R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


71 type of off-flavor generated clearly depends on the storage conditions (e.g. light, temperature, storage time). Up to now, more than 630 volatile compounds have been identified in various types of beer (5). Among them short-chain fatty acids, carbonyl compounds and esters (in total 330 compounds) predominate. Within the past five decades different compounds have been suggested as the cause for the development of less desired aromas in beer. Burger et al. (6, 7) have suggested acetaldehyde and furfural to generate cardboard-like aromas in beer, when added in concentrations of 30 mg/L or 2 mg/L, respectively. These findings, however, could not be confirmed in later studies (8, 9). In addition, (E)β-damascenone (10) and 2-aminoacetophenone (11) have been proposed as further contributors to beer staling and also the Strecker aldehydes 2methylpropanal as well as 2- and 3-methylbutanal have been suggested to increase the malty, cereal-like aromas in beer during storage (12). Furthermore, 4methyl-4-mercaptopentan-2-one was identified as cause of a ribes-type off-flavor (13), and another sulfur compound, 3-methyl-2-butene-l -thiol, is long-known as the cause for the so-called "sunstruck" off-flavor in beer (14). As mentioned above, oxygen has longbeen suggested as an important factor reducing beer stability. Based on the observation that the redox potential in beer decreases during aging, De Clerck (75) already sixty-eight years ago has proposed this crucial role of oxygen in beer flavor degradation. Because oxygen is an important substrate in lipid peroxidation, many studies have focused on this parameter. Jamieson and Gheluwe (8) identified (E)-2-nonenal, a well known degradation product of unsaturated fatty acids, in beer and have proved by means of sensory experiments that concentrations in the ppb-range were able to generate a cardboard-like off-flavor. The odor threshold of (E)-2-nonenal in beer lies between 0.04 and 0.5 μg/L (cf. review by Collin (16)). However, its role in beer off-flavor is not yet clear, because some authors found increased amounts of this aldehyde in stored beers (17, 18,) while others did not find an increase (19, 20). B y application of Aroma Extract Dilution Analysis, a method based on sniffing of G C eluates (21), we could previously show that the Flavor Dilution (FD) factors of, in particular, phenylacetaldehyde, 3-methyl-3-mercaptobutylformate, (E,E)-2,4-nonadienal, (E)-2-nonenal and an unknown compound with an aniseed-like odor quality were increased in a forced aged beer (14 days at 37°C) compared to the respective fresh beer (22). Evans et al. (23), applying GC/Olfactometry, later on confirmed the role of phenylacetaldehyde in beer staling and suggested methional and 4-methoxybenzaldehyde as further off-flavor contributors. This brief literature survey shows that it is not yet clear, which compounds can be regarded as the main contributors to beer staling. There is, therefore, no reliable method available to objectify flavor changes occurring during beer storage. Several reasons can be given for this lack in information: (i) Depending on the storage parameters different off-odorants may be generated, (ii) the methods of quantitation applied in the older studies, e.g. derivatization of carbonyls with dinitrophenyl hydrazine, may result in degradation of precursors by the conditions applied in the derivatization procedure, (iii) a combination of sensory and analytical methods, e.g. the Aroma Extract Dilution Analysis, has


72 only been applied in very few studies and (iv) most of the studies have used forced aged beers by application of a thermal treatment and/or an oxygen administration. The objectives of the present study were, therefore, to show the contribution of single odorants to beer flavor aging by application of a comparative Aroma Extract Dilution Analysis on a fresh and a naturally aged beer, and by subsequent identification and quantitation of the most odor-active compounds.


Materials and methods The beer samples (pale lager beer) were supplied by a Dutch brewery. Storage conditions applied were 20°C in the original bottle, i.e. in the dark (naturally aged beer: N A B ) . For the isolation of the volatiles, the beer sample (500 mL) was extracted with diethyl ether and the volatile compounds were separated from the non-volatile material by a SAFE-distillation (24). Further separation steps and the application of the Aroma Extract Dilution Analysis were performed as described elsewhere (25). The most important odorants were quantified by stable isotope dilution assays (SIDA) (25). For the quantitation of (E)-2-nonenal a very selective and sensitive procedure of coupling the SIDA with high resolution mass spectrometry was used (26).

Results Odorants in a fresh pale lager beer The volatile compounds in a fresh pale lager beer were isolated by extraction with diethyl ether followed by high vacuum distillation. Application of the Aroma Extract Dilution Analysis on the concentrated extract (500 m L beer ~> 100 of extract; concentration factor 1:5000) revealed a total of 23 odor-active compounds in the fraction containing the neutral and basic volatile compounds and an additional number of 18 odorants in the fraction containing the acidic volatile compounds (25). In the Flavor Dilution (FD) factor range of 16 to 8192 thirty-eight odorants were identified. Among them, the ten aroma compound displayed in Fig. 1 showed the highest FD-factors. The results revealed, especially 2-phenylethanol, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 3-hydroxy4,5-dimethyl-2(5H)-furanone and (E)-B-damascenone as the most odor-active compounds in the aroma of the fresh beer, thereby confirming data of our previous study (22). With the exception of ethyl3-phenylpropanoate (FD 1024) and 2,3-dihydro5-hydroxy-6-methyl-4(H)-pyran-4-one (FD 64) all other compounds have previously been reported in the literature as beer volatiles although their flavor contribution had not been studied.


73

Λ Ι

.

HO

Ο

JCL (flowery; 8192)

(caramel-like; 4096)

\

OH

J&. (seasoning-like; 4096)

(cooked apple; 2048)

OH Q

ι

JL -OCH,

^ - ^ O H


(malty; 1024)

(fruity; 1024)

(clove-like; 1024)

(fruity; 1024)

J (honey-like; 1024)

(fruity; 1024)

Figure 1. Structures of ten key odorants identified in fresh pale lager beer after application of an Aroma Extract Dilution Analysis (Odor quality, FD-factor) Key odorants in naturally aged beer Beer samples of the same batch were stored for a maximum of 34 months at 20°C in the dark in the original brown glass bottles. Every month, an overall sensory evaluation was performed by means of the triangle test and by flavor profile analysis. Fresh beer of the same type was supplied by the brewery every 2 months for comparison. In general, even after 34 months, the beer was drinkable, but significant differences in the overall flavor were observed after about 12 months. The flavor impression clearly shifted to a more sweet-malty, honey- and cider-like aroma with a decrease in the hoppy, flowery aroma. Application of A E D A on a flavor extract isolated from the beer stored for 34 months at 20°C in the dark revealed the same odorants as identified in the fresh sample. The FD-factors of most of the odor-active odorants (cf. Fig. 1) were nearly identical with the FD-factors determined in the fresh sample indicating that neither a decomposition nor a formation of these odorants occurred during storage. B y contrast, in particular four compounds, namely ethyl 2-methylpropanoate (fruity), ethyl 2-methylbutanoate (fruity), 3-(methylthio)propanal (potato-like) and phenylacetaldehyde (sweet, honey-like) were significantly increased during storage (Table 1).


74 Table 1. Important odorants showing clear differences in their Flavor Dilution (FD) factors in naturally aged beer (NAB) compared to fresh beer

m

Odorant

Odor quality

Ethyl 2-methylpropanoate Ethyl 2-methylbutanoate 3-(Methylthio)propanal Phenylacetaldehyde

Fruity Fruity Potato-like Honey-like

FDin NAB 1024 64 128 128

FB 64 16 16 16


Quantitation of selected odorants The data given above have shown that the flavor changes occurring during natural beer aging are not caused by formation of new aroma compounds, but by an increase of odorants already present in the fresh beer. To study such changes in more detail, twenty-seven of the odorants were quantified in the fresh and the naturally aged beer by application of stable isotope dilution assays (25). As already suggested by the results of the A E D A , most of the odorants identified as key odorants in the fresh beer (cf. Fig. 1) were not much influenced by the storage (Table 2). Although their concentrations differed slightly in both samples, their "relative odor activity value" (ratio of concentration to odor threshold) was only by a factor of 1.5 higher in the stored sample, e.g. for 4hydroxy-2,5-dimethyl-3(2H)-furanone.

Table 2. Comparison of the concentrations of five important beer odorants in the naturally aged (NAB) and in the fresh beer (FB) Cone. (μ&Ι) Odorant — NAB FB 49300 3-Methylbutanol 54000 39100 2-Phenylethanol 41400 427 314 Phenylacetic acid 284 4-Hydroxy-2,5-dimethyl-3(2H)-fiiranone 170 72 4-Vinyl-2-methoxyphenol 86

However, a completely different result was obtained for the six compounds given in Table 3. In particular, phenylacetaldehyde and 2-methylpropanal were increased by a factor of five, and ethyl 3-methylbutanoate showed a more than ten times higher concentration in the stored sample.


75 Quantitative experiments aimed at determining the time course of the formation of these odorants during storage revealed that ethyl 2-methylbutanoate (Fig. 2) and also phenylacetaldehyde (Fig. 3) clearly increased with storage time. Consequently, both compounds can be suggested as indicator aroma compounds to assess the flavor stability of beer.

Table 3. Comparison of the concentrations of six aroma compounds, which were clearly increased in naturally aged beer (NAB) compared to fresh beer


m C

Odorant 2-Methylpropanal 3-Methylbutanal Methional Phenylacetaldehyde Ethyl 2-methylpropanoate Ethyl 3-methylbutanoate

ο

m

a

FB 11 12 1 5 67 20

θ4

°

0

— NAB 62 32 3 25 367 233

1

1

1

1

10

2D

30

40

time [months] Figure 2. Time course of the formation of ethyl 2-methylpropanoate in beer during "natural" aging at 20°C in the dark

The role of (E)-2-Nonenal As detailed in the "Introduction", (E)-2-nonenal has been suggested many times as the key "off-flavor" compound in beer, although the role of this aldehyde in beer staling is discussed quite controversely. A s shown by other groups and confirmed also in the author's own studies (19, 26), by addition of 1 μg/L of (E)2-nonenal to fresh beer, a cardboard-like off-flavor is generated. The crucial question, however, is whether enough (E)-2-nonenal is generated during storage to exceed this "breakthrough" value. B y definition, this is the threshold at which a


76


single compound can clearly influence a food aroma in the presence of the whole set of food volatiles.

Figure 3. Time course of the formation of methional and phenylacetaldehyde during natural beer aging

It is a challenge in the analysis of such aroma-active compounds occurring in the sub-ppb level to get reliable quantitative data. In many of the studies published, no internal standard has been used. Furthermore, to increase sensitivity, very often derivatization procedures are used in which the application of heat and or low p H values is necessary. Such procedures, however, may generate (E)-2-nonenal from precursors present in the beer or extract, respectively, thereby yielding higher amounts than originally present in the beer. Stable isotope dilution assays using internal standards labeled with either Deuterium or Carbon 13 (100 % labeling) have been successfully applied in the analysis of aroma compounds which are unstable and/or occur in trace amounts (21). Using [ H ]-(E)-2-nonenal as the internal standard, two different isotope dilution assays were developed and applied to quantify this odorant in beer (26). One is based on the enrichment with two dimensional gas chromatography before mass spectral measurement, the other uses a direct measurement by means of high resolution mass spectrometry (26). For the latter approach, a beer sample (1200 mL) was spiked with [ H ]-(E)-2-nonenal (0.2 μg) and, after isolation of die volatile fraction, directly analyzed by HRGC/high resolution mass spectrometry. As indicated in Fig. 4, monitoring of the respective mass of the labeled and the unlabeled aldehyde allowed an exact quantitation directly from the extract without any enrichment step. B y application of this method, the concentration of (E)-2-nonenal could reliably be followed during storage. The results summarized in Table 4 clearly showed that there is no correlation in (E)-2-nonenal concentration with time, because even higher amounts were detected in the 1 month old beer compared to a beer stored for 34 month. In no case, the breakthrough threshold of 0.1 μg/L was matched. The data were in good agreement with the results of the sensory evaluations, because in no sample a cardboard-like off-flavor could be detected sensorially. The data clearly 2

2

2

2


77 reveal that in the beer type investigated, (E)-2-nonenal is not a contributor to the flavor changes occurring during storage.

%

rn/z = 83.0496

100

A = 6737

soi 0

m/z = 85.0612 Downloaded by EMORY UNIV on February 4, 2016 | http://pubs.acs.org Publication Date: October 10, 2002 | doi: 10.1021/bk-2003-0836.ch005

loo ι A = 12089

50 0

loo -f so 1 1

1 '

11:30

11:50

time (min)

Figure 4. High resolution gas chromatography/high resolution mass chromatography of (E)-2~nonenal and [ HJ'(E)~2-nonenal Quantitation in a naturally aged beer (23 months, 20°C) containing 0.07 μg (E)-2-nonenalperL 2

a

Table 4. Concentrations of (E)-2-nonenal determined by a stable isotope dilution assay in naturally aged beer

a

b

Storage time (months) 0 1 4 12 23 34 The breakthrough threshold was determined to be 0.1 Limit of confidence ± 10 %.

Cone. (μg/L) 0.01 0.05 0.02 0.03 0.07 0,04

Conclusions The results confirm that significant flavor changes do occur even i f a beer is stored in the dark at room temperature. (E)-2-Nonenal was clearly excluded as an


78 important contributor to the sweet, cider- and honey-like flavor of the aged beer, but an increase in the Strecker aldehydes and in some esters clearly reflected this flavor change. Flavor recombination studies and dotation experiments using reference odorants are underway to prove the increase in Strecker aldehydes and esters as crucial for beer flavor stability. Preliminary studies already revealed that the classical way of Strecker aldehyde formation (27) does not apply in the formation pathway of these aldehydes in beer, but that other mechanisms are operating to generate these off-odorants during storage.


References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Drost, B . W . ; Van Eerde, P.; Hoeckstra, S.; Strating, J. Proc. Eur. Brew. Conv. 1971, pp. 451-458. Meilgaard, M . C . Brew. Dig. 1972, 47, 48-57. Dagliesh, C . A . Proc. Eur. Brew. Conv. 1977, 623-659. Clapperton, J.F. J. Inst. Brew. 1976, 82, 175-176. Nijssen, L .M.; Visscher, C.A.; Maarse, H .; Willemsen, L . C . ; Boelens, M . H . In: Volatile compounds in food. Qualitative and quantitative data, supplement 6, 7th ed., T N O Nutrition and Food Research, Zeist, The Netherlands, 1996. Burger, M .; Glenister, P.; Becker, K . Proc. Am. Brew. Chem, 1954, 98-107. Burger, M. In: Brewing Science, Part 2, Pollock, J.R.A.; ed.; Academic press, London, 1959, pp. 348-405. Jamieson, A .M.; Van Gheluwe, J.E.A. Proc. Am. Soc. Brew. Chem. 1970, 123-126. Ahrenst-Larsen, B . ; Levin-Hansen, H . Monatsschr. Brauwiss. 1963, 16, 393-397. Strating, J.; van der Eerde, P. J. Inst. Brew. 1973, 79, 414-415. Palamand, S.R.; Grigsby, J.H. Brew. Dig. Sept. 1974, 58-59, 90. Kossa, T.; Bahri, D.; Tressl, R. Monatsschr. Brauwiss. 1979, 32, 249-252. Cosser, K . B . ; Murray, J.P.; Hozapfel, C . W . Tech. Q. Master Brew. Assoc. Am. 1980, 77, 53-59. Kuroiwa, Y .; Hashimoto, N . Proc. Am. Soc. Brew. Chem. 1961, 181-19. De Clerck, J. J. Inst. Brew. 1934, 40, 407-419. Collin, S.; Noel, S. Cerevisia Biotechnol 1994, 19, 25-32. Sakuma, S.; Kowaka, M. J. Am. Soc. Brew. Chem. 1994, 52, 37-41. Lermusieu, G.; Noel, S.; Liegeois, C.S. J. Am. Soc. Brew. Chem. 1999, 57, 29-33. Schieberle, P. In: Aroma production and application, Deutsches Institut für Ernährungsforschung Potsdam, Rothe, M .; ed.; 1992, pp. 137-154. Lustig, S. Ph.D. Thesis, Technical University of Munich, 1994. Schieberle, P. In: Characterization of Food: Emerging Methods; Goankar, A . D . ; ed.; Elsevie Science B . B . , 1995, pp. 403-431. Schieberle, P. Z. Lebensm. (Inters. Forsch. 1991, 193, 558-565.


79 Evans, D.J.; Schmedding, D.J.M.; Bruinje, Α.; Heideman, T.; King, B .M.; Groesbeek, N .M. J. Inst. Brew. 1999, 5, 301-307. 24. Engel, W.; Bahr, W.; Schieberle, P. Eur. Food Res. Technol. 1999, 209, 237-241. 25. Komarek, D.; Schieberle, P. Eur. Food Res. Technol. 2002, submitted. 26. Komarek, D.; Schieberle, P. J. Agric. Food Chem. 2002, submitted. 27. Hofmann, T.; Schieberle, P. J. Agric. Food Chem. 2000, 48, 4301-4305.


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Changes in Key Aroma Compounds during Natural Beer Aging

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