Comparison of (+)Catechin and Ferulic Acid as Natural Antioxidants and Their Impact on Beer Flavor Stability.

Part 2: Extended Storage Trials

M. T. Walters,1 A. P. Heasman, and P. S. Hughes, BRF International, Lyttel Hall, Nutfield, Surrey, RH1 4HY UK
ABSTRACT J. Am. Soc. Brew. Chem. 55(3):91-98, 1997 During extended storage at 0, 25, or 40C, there were measurable changes in the concentration of some carbonyl compounds, UV-active compounds separated by reverse-phase HPLC, and iso--acids. The rate of change increased markedly with temperature. The antioxidants (+) catechin and ferulic acid had no impact on the rate of formation of carbonyl compounds but did change the rate of change of some UV-active compounds and the cis-iso--acids. In contrast, (+)catechin and ferulic acid altered the rate of formation of carbonyl compounds during forcedaging. Beer forced-aged in the absence of air produced a carbonyl pattern similar to that produced during extended storage. Additional carbonyl compounds were formed during exposure to air at high temperatures, and it is the formation of these compounds that was affected by the presence of (+)catechin or ferulic acid. It appeared that two mechanisms of carbonyl formation were in operation during storage. The first occurred at low levels of oxygen (<0.1 ppm) and involved the formation of trans-2nonenal. The second occurred on exposure to air, producing additional long-chain carbonyl compounds, including undecanal. The antioxidants (+)catechin and ferulic acid affected carbonyl formation by the second mechanism but not the first. Keywords: Antioxidants, Carbonyl compounds, (+)Catechin, Ferulic acid, Iso--acids, Storage trials RESUMEN Durante almacenamiento prolongado a 0, 25, o 40C hubo cambios medibles en la concentracin de algunos compuestos carbonilos, compuestos activos al UV separados por HPLC fase reversa e iso--cidos. La velocidad del cambio aumento marcadamente con la temperatura. Los antioxidantes (+)catequina y cido ferlico no tuvieron impacto en la velocidad de formacin de compuestos carbonilos pero si en los cambios de algunos compuestos activos al UV y cis-iso--cidos. En contraste, se encontr que la (+)catequina y el cido ferlico alteran la proporcin de formacin de compuestos carbonilos durante el envejecimiento forzado. Cervezas sometidas a envejecimiento forzado en ausencia de aire produjeron un patrn de carbonilos similar al producido durante almacenamiento prolongado. Compuestos carbonilos adicionales fueron formados durante exposicin al aire a altas temperaturas y es la formacin de estos compuestos los que fueron afectados por la presencia de (+)catequina cido ferlico. Parece que dos mecanismos de formacin de compuestos carbonilos estuvieron en operacin durante el almacenamiento. El primero ocurri a niveles bajos de oxigeno (<0.1 ppm) e involucra la formacin de trans-2 nonenal. El segundo ocurri en la exposicin al aire, produciendo compuestos carbonilos de cadena larga, incluyendo undecanal. Los antioxidantes (+)catequina y cido ferlico afectan la formacin de carbonilo por el segundo mecanismo, no por el primero.

catechin and concentrations of <11 mg/L for ferulic acid (10). Polyphenols and phenolic acids in beer have an impact on quality issues such as flavor and colloidal stability. In particular, the amount of (+)catechin in beer has been linked to haze formation by oxidative polymerization (1). The addition of (+)catechin and ferulic acid to beer resulted in significant chemical changes in beer composition following forced aging at 60C for 24 hr. (+)Catechin decreased the rate of formation of some carbonyl compounds including the long-chain unsaturated aldehyde, undecanal. In contrast, ferulic acid increased the rate of formation of some other carbonyl compounds. Changes in UV-active compounds separated by reverse-phase (RP) HPLC were also observed and correlated with (+)catechin and ferulic acid levels in the beer. (+)Catechin and ferulic acid were found to be acting independently (12). This work is consistent with and expands on previous findings by Dadic et al (3), where total carbonyls were measured in beer stored with low and high levels of air. (+)Catechin decreased the rate of total carbonyl formation in the high-air beers but not in the low-air beers. Ferulic acid increased the rate of carbonyl formation in both low-air and high-air beers. In this study, (+)catechin and ferulic acid were added to beer before filtration and packaging, and chemical markers of aging were monitored during extended storage trials. A summary of the results obtained on the effect of storage conditions on carbonyl formation and the impact of air on carbonyl formation has been published elsewhere (13). MATERIALS AND METHODS Packaging of Beer A lager-style beer was collected in three 25-L kegs and dosed with (+)catechin (1 mM final concentration), ferulic acid (1 mM final concentration), or 20% ethanol (v/v) (the solvent used for additions) as a blank. The (+)catechin and ferulic acid solutions were prepared under nitrogen in 500 ml of 20% ethanol (v/v) to aid solubility. The solutions were dosed into beer under nitrogen to prevent oxygen pickup. Beer was bottled from the keg using the pilot brewing facility at BRFI and pasteurized (20 PU). The bottled beer (275 ml) was stored at 0, 25, or 40C before analysis. The effect of pasteurization and antioxidant addition was tested using two balanced experimental designs and two-way analysis of variance, one for (+)catechin addition, the other for ferulic acid addition. Data was analyzed using STATISTICA 5.0 (StatSoft, Tulsa, OK). Five bottles of beer were used for each combination of variables. There were two levels of each variable, antioxidant (0 or 1 mM) and pasteurization (pasteurized or unpasteurized). Each sample was analyzed for carbonyl compounds by gas chromatography with electron capture detection (GC-ECD) (11). Measuring Oxygen Content The oxygen content of the bottled beer was measured using a beverage package sampler (model 29972, Orbisphere Laboratories). Twenty-four bottles packaged from the pilot brewery and five commercially packaged bottles from the same batch were measured. 91

(+)Catechin, a polyphenol, and ferulic acid, a phenolic acid, have shown useful antioxidant properties based on model assay systems (5,9,11,12). These compounds are present in malt and have been detected in beer in concentrations of <15 mg/L for (+)
1 Corresponding

author. Phone: +44(0)1737 822 272. Fax: +44(0)1737 822 747.

Publication no. J-1997-0621-01R. 1997 American Society of Brewing Chemists, Inc.

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Walters, M. T., Heasman, A. P., and Hughes, P. S. slowly bubbled into the bottle or test tube for 1 min. Each combination of variables was set up in triplicate. The beer was heated to 60C for 22 hr or stored at 0C for 22 hr before analysis using the GC-ECD method described in Walters et al (11). Effect of Temperature on Formation of Carbonyl Compounds Fresh beer (6.0 ml) and beer stored at 0C for 97 days (6.0 ml) were transferred to sealed glass vials and stored at 0, 25, 40, 50, or 60C for 24 hr. A 1-ml aliquot was analyzed for UV-active compounds separated by RP-HPLC. The 5.0-ml sample was analyzed for carbonyl compounds by GC-ECD (11). RESULTS AND DISCUSSION Storage of Dosed Beer The impact of (+)-catechin (1 mM) and ferulic acid (1 mM) on carbonyl formation was tested in beer dosed before packaging and stored at 0, 25, or 40C for up to five months. Beer was dosed with either (+)catechin, ferulic acid, or 20% ethanol under nitrogen before bottling to prevent air ingress. After packaging, the oxygen content of 24 control bottles was measured using an Orbisphere oxygen meter. The level of dissolved oxygen in the dosed control beer (mean = 0.058 ppm, standard deviation = 0.021 ppm, n = 24) was similar to that in commercially packaged beer (mean = 0.066 ppm, standard deviation = 0.034 ppm, n = 5) that had been collected at the same time as the kegged beer and stored at 0C. After bottling, a carbonyl profile was obtained before and after pasteurization. A number of peaks showed a small but significant (P < 0.05) decrease in peak area following pasteurization when tested by two-way analysis of variance with pasteurization and antioxidant addition as the two variables. An example of a GC chromatogram with a tentative identification of some peaks is shown in Walters et al (12). One compound (peak 4) increased in concentration after pasteurization in both experiments. When the

HPLC Analysis An aliquot of beer was collected at each time point and analyzed for UV-active compounds separated by RP-HPLC according to the method described in Walters et al (12). Iso--Acids The iso--acids (cis- and trans-isohumulone, cis- and transisocohumulone, cis- and trans-isoadhumulone) were separated by RP-HPLC using isocratic elution. Samples of freeze-dried beer (200 mg) that had been stored at 20C were dissolved in 10 ml of distilled water before analysis. Beer (100 ml) produced 2.7 g of freeze-dried solid. Samples (20 l) were injected using a Rheodyne 7125 HPLC injector on to a Novopak C18 (4 m) radial compression cartridge enclosed in a Waters RCM-100 module. The column was equilibrated in ultrafiltered solvent consisting of acetonitrile, water, methanol, formic acid, trifluoracetic acid, and aqueous 0.2M magnesium acetate (840:490:480:3.6:1.2:24.0, v/v) and run isocratically at 2 ml/min using a Waters chromatography pump and Waters UV-detector model 440 set at 280 nm (7). Using this methodology, cis-isohumulone and trans-isoadhumulone coelute. Nevertheless, as trans-isoadhumulone is a minor iso-acid in beer, the total area of this peak is 90% cis-isohumulone. A 50 mg/L trans-iso--acids standard as the dicyclohexylamine salt in 96%(v/v) ethanol was used to calculate the concentration of iso--acids in the freeze-dried beer samples. Total Carbonyl Analysis Samples of beer (5 ml) were transferred into 20-ml crimp-top vials and 2 ml of 0.33% (v/v) thiobarbituric acid in 50% glacial acetic acid and H2O (v/v) were added. The vials were crimp-capped and incubated in a heated waterbath set at 60C for 1 hr. The samples were rapidly cooled. Absorbance was measured at 530 nm. Samples were taken at various time points during the storage of the beer. A duplicate sample was taken from each bottle. Carbonyl Analysis An aliquot of beer was collected at each time point and was analyzed for carbonyl compounds as described in Walters et al (11). Comparing Forced-Aging in the Presence and Absence of Air The effect of aeration on carbonyl formation in packaged beer was tested using two full-factorial design experiments with two-way analysis of variance using STATISTICA 5.0. Both experiments involved two variables: temperature and aeration. The first experiment was conducted using bottled beer, the second using sealed vials as in the forced-aging test (12). The temperature was held at 0 or 60C, and samples contained either no air or air that had been

Fig. 1. Rate of increase for peak with a retention time of 20.2 min in beer dosed with 1 mM ferulic acid stored at 0 ( ), 25 ( ), or 40C ( ).

Fig. 2. Comparison of the UV-active compounds separated by reversephase HPLC for beer stored at 0C (A) and at 40C (B) for 156 days. Retention time (min) for peaks are 1: <4.5; 2: 6.03; 3: 6.59; 4: 7.15; 5: 8.10; 6: 9.77; 7: 10.25 (+)catechin; 8: 11.06; 9: 12.14; 10: 13.22; 11: 13.81 ferulic acid; 12: 14.12; 13: 20.17 4-vinylguaiacol.

(+)Catechin and Ferulic Acid as Natural Antioxidants. 2: Extended Storage Trials data was pooled for the ferulic acid and (+)catechin treatments and the effect of pasteurization was determined by a Students t-test, there were only two peaks (3-methyl-2-butenal and peak 12) showing a significant (P < 0.05) decrease upon pasteurization. It would appear from the data as a whole that pasteurization had little impact on the profile of carbonyl compounds with any effect being to slightly reduce the level of a few of the carbonyl peaks. Grnqvist et al (6) found only small differences in carbonyl compounds following pasteurization. The peak with a retention time of 33.0 min increased in concentration with the addition of ferulic acid, which was also observed in the forced-aging study (12). Measuring UV-Active Components in Dosed Beer by RP-HPLC During Storage Both (+)catechin and ferulic acid were separated and detected by RP-HPLC using UV detection at 280 nm. Solutions of 10 mM (+)catechin and 10 mM ferulic acid were run as blanks. The blank (+)catechin solution had three peaks with retention times of 9.77, 10.25, and 11.06 min. The largest peak was at 10.25 min (94% by area at 280 nm). The control beer had a peak at 11.06 min. The blank ferulic acid solution had three peaks with retention times of 13.81, 14.33, and 20.17 min. The largest peak was at 13.81 min (96% by area at 280 nm). The control beer had no peaks with these retention times. The concentrations of ferulic acid and (+)catechin in the control beer were below the detection limits of the method. The detection limits of the method were 5 M for (+)catechin and 5 mM for ferulic acid.

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The major (+)catechin and ferulic acid peaks decreased during storage and this was temperature-dependent, with the greatest rate of loss occurring at 40C. There was a minimal decrease in ferulic acid concentration for beers stored at 0 or 25C. In contrast, there was a large decrease in (+)catechin concentration for beers stored at 25 or 40C, with the greatest loss occurring at 40C. There was a greater loss of ferulic acid (36% at 40C) than (+)catechin (26% at 40C) after 160 days of storage. The peak with retention time of 20.17 min increased in concentration during storage in the beer dosed with ferulic acid only (Fig. 1). This was identified as 4-vinylguaiacol based on retention time and UV spectra of a standard solution obtained using a diode-array detector (12). The presence of this compound in beer produces a strong clove-like flavor and aroma, and would be considered a negative attribute for most beers (2). The rate of formation of 4-vinylguaiacol was strongly temperature-dependent. It took 10 days to appear in beer stored at 40C and 70 days to appear in beer stored at 25C. It was absent in beer stored at 0C for up to 160 days. It was also absent in the control and (+)catechin dosed beers, and it was most likely formed as a thermal breakdown product of ferulic acid. None of the other minor peaks changed during storage. Comparison of the control beer stored at 40C with beer stored at 0C revealed some large changes in the UV-active compounds (Fig. 2). The major changes were an increase in peaks with a retention time of 8.10 min and a decrease in peaks with a retention time of 6.03 min. There was also a smaller decrease in the amount of compounds with retention times of 6.59 and 7.15 min during storage.

Fig. 3. Rate of change for peak with a retention time of 8.10 min. A, Control beer stored at 0 ( ), 25 ( ), or 40C ( ). B, Beer dosed with 1 mM (+)catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C.

Fig. 4. Rate of change for peak with a retention time of 6.03 min. A, Control beer stored at 0 ( ), 25 ( ), or 40C ( ). B, Beer dosed with 1 mM (+)catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C.

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Walters, M. T., Heasman, A. P., and Hughes, P. S. age. The presence of ferulic acid at a concentration of 1 mM, and to a lesser extent (+)catechin at 1 mM, in the beer reduced the rate of loss at 40C of the iso--acids. There was a rapid drop in concentration of the trans-isomers for beer stored at 40C and a much slower drop for beers stored at 25C. Beers stored at 0C did not show a decrease in concentration after 220 days of storage. The rate of decrease was similar for trans-isohumulone and trans-isocohumulone with trans-isohumulone being undetectable in the beer after 120 days of storage at 40C, and trans-isocohumulone reaching a minimum concentration of 0.3 mg/L after a similar length of time (Fig. 5). The presence of (+)catechin (1 mM) and ferulic acid (1 mM) did not significantly affect the rate of loss of the trans-isomers. In contrast, there was a much slower rate of loss of the cis-isomers when compared with loss of trans-isomers at 40C. There was no loss of cis-isomers in beers stored at 0 or 25C. The presence of 1 mM ferulic acid decreased the rate of loss for cis-isoadhumulone, cis-isohumulone, and cis-isocohumulone. The addition of 1 mM (+)catechin also delayed the loss of the three cis-isomers but was less effective than ferulic acid (Fig. 6). During the extended storage trials there was a large drop in the concentration of iso--acids that was strongly temperature-dependent. The trans-isomers were lost at a rapid rate as compared to the loss of cis-isomers. The presence of ferulic acid (1 mM) or (+)catechin (1 mM) did not alter the rate of loss of the transisomers but reduced the rate of loss of the cis-isomers. Ferulic acid was more effective than (+)catechin. The major loss of iso-acids occurred in beer that had been stored at 40C for up to five months. Carbonyl Formation in Dosed Beer During Storage The addition of 1 mM (+)catechin to beer decreased total carbonyl development after storage at 40C for a period of up to 160 days (Fig. 7). There was no difference in the total carbonyls for the treated beers stored at 0 and 25C for up to 160 days. Storage at

The peak with a retention time of 8.10 min increased markedly during storage at 40C but not at 0 and 25C. Its rate of increase was influenced by (+)catechin and ferulic acid (Fig. 3). Ferulic acid increased the rate of formation, whereas (+)catechin decreased the rate of formation relative to the control. The rate of decrease of peak 6.03 was reduced in the presence of ferulic acid and (+)catechin with the effect being greater for ferulic acid at 40C (Fig. 4). The effect was also noted at 25C for peak 6.03, but it was of reduced magnitude. The rate of decrease of peaks 6.59 and 7.15 was not as substantially affected by (+)catechin and ferulic acid, but it was slightly reduced in the presence of these antioxidants. The rate of change was far greater at 40C for all of these compounds. It is clear from this work and from forced-aging studies (12) that UV-active compounds change in concentration when beer is stored at elevated temperatures. The presence of (+)catechin and ferulic acid affects the rate of these changes during both extended storage and forced-aging (12). Degradation of Iso--Acids During Storage Organoleptic criteria show a decrease in bitter taste during aging of beer (4). The main bittering compounds present in the beers under investigation were the iso--acids (cis- and trans-isohumulone, cis- and trans-isocohumulone, and cis- and trans-isoadhumulone). The concentration of these compounds was measured during the extended storage trials by HPLC. There was a negligible loss of iso--acids during the freeze-drying process. The total iso--acid content of bottled beer was 15.7 mg/L compared with 15.2 mg/L for beer from the same batch that had been freezedried. The initial total concentration of iso--acids in the beer used for the extended storage trials was 15.3 mg/L. After storage at 40C for 156 days, the concentration in the control beer was reduced to 4.5 mg/L (71% decrease). In the beer dosed with (+)catechin, the concentration was reduced to 6.5 mg/L (58% decrease), while in the beer dosed with ferulic acid, the concentration was reduced to 8.2 mg/L (46% decrease). There was no loss of total iso--acids in beers stored at 0 and 25C after 220 days of stor-

Fig. 5. Rate of change in beer. A (trans-isohumulone) and B (transisocohumulone): Beer stored at 0 ( ), 25 ( ), or 40C ( ). C (transisohumulone) and D (trans-isocohumulone): Beer dosed with 1 mM (+) catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C.

Fig. 6. Rate of change in beer. A (cis-isohumulone and transisoadhumulone), B (cis-isocohumulone), and C (cis-isoadhumulone): Beer stored at 0 ( ), 25 ( ), or 40C ( ). D (cis-isohumulone and transisoadhumulone), E (cis-isocohumulone), and F (cis-isoadhumulone): Beer dosed with 1 mM (+)catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C.

8 6

(+)Catechin and Ferulic Acid as Natural Antioxidants. 2: Extended Storage Trials these temperatures did not result in an increase in total carbonyls after 220 days of storage, whereas there was a notable increase in total carbonyls for beers stored at 40C. The beers were exposed to air before analysis of total carbonyls and heated to 60C during the analysis and, as a result, additional carbonyl compounds may have been formed due to this exposure to air. This result is consistent with the results obtained from the forced-aging tests in which beer was also exposed to air during the forcing period (11,12). The carbonyl profile was also measured for the bottled beers stored at 0, 25, or 40C by GC-ECD. In contrast to the results obtained by forced-aging (11,12) and the total carbonyl assay, there was no difference in the rate of formation of any carbonyl compounds measured for the beers treated with 1 mM (+)catechin or 1 mM ferulic acid and stored in bottles at 0, 25, or 40C. The majority of carbonyl compounds (peaks 13, 5, 6, 8, 1113, 1622, and 24) did not respond to heat or storage time. Some of these peaks have been tentatively identified based on retention time as formaldehyde, acetaldehyde, acetone, 3-methyl-2-butenal, trans-2-octenal, and undecanal. There was some evidence of a slight decrease in concentration for acetaldehyde during storage, which is consistent with large molecular weight carbonyls being formed by aldol condensation reactions. Another group of compounds (peaks 4, 7, 9, 10, 14, and 15) increased with storage time and heating and included compounds such as furfural, hexanal, trans-2-nonenal, and 5-hydroxymethylfurfural (Fig. 8). An increase in hexanal and furfural has been noted by others (6,8) during ex-

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tended storage and forced-aging. One compound (peak 23) increased with the addition of ferulic acid, which was also observed in the forced tests. Peak 25 was not detected in these beers, but it was present in the forced-aged beers treated with ferulic acid. Comparison of Carbonyl Formation During Forced-Aging and Extended Storage Trials The carbonyl profile for beer stored at 40C during the storage trial was different from that obtained from the forced-aging experiments (Fig. 9). A major difference was that in the forced tests, the beer was exposed to air, whereas in the storage trial, exposure to air was minimized. An experiment was designed to investigate whether the addition of air to bottled beer could explain the observed results. Two balanced designed experiments were implemented with two levels of air and temperature for bottled beer and beer transferred to sealed vials and exposed to air (forced-aging) (12). Each combination was measured in triplicate. Three groups of peaks could be identified based on their response to change in temperature and exposure to air. The first group (peaks 2, 3, 5, 11, 12, and 24) did not respond to heat or air (Tables I and II). This group also did not change during the storage trial. A second group (peaks 1, 4, 7, 8, 9, 10, 14, and 15) changed with heat but not air (Tables III and IV). These peaks (except for peaks 1 and 8) increased during the storage trial and increased at a greater rate with higher temperature. The third group (peaks 6, 13, 1623) responded to a combination of heat and air (Tables V and VI). These compounds did not change during the storage trial and were present in the beer during the storage trial at low levels. Heating during storage at 40C for 120 days or heating to 60C for 24 hr did not result in an increase in these peaks. The addition of air to bottled beer before heating to 60C resulted in a significant (P < 0.05) increase in the relative peak area of these compounds. The addition of air to the glass vial had no impact, as the beer in the vials had been exposed to air during transfer from the bottle (Tables V and VI).

Fig. 7. Rate of total carbonyl change. A, Control beer stored at 0 ( ), 25 ( ), or 40C ( ). B, Beer dosed with 1 mM (+)catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C.

Fig. 8. Rate of change in beer. A (furfural), B (trans-2-nonenal), and C (5-hydroxymethylfurfural): Beer stored at 0 ( ), 25 ( ), or 40C ( ). D (furfural), E (trans-2-nonenal), and F (5-hydroxymethylfurfural): Beer dosed with 1 mM (+)catechin ( ), 1 mM ferulic acid ( ), and control ( ) stored at 40C. Some data published previously (13).

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Walters, M. T., Heasman, A. P., and Hughes, P. S. whereas the second involves storage time, heat, and exposure to air. The addition of (+)catechin and ferulic acid to beer during forced-aging altered the rate of formation of carbonyl compounds formed predominantly by this second mechanism. The addition of (+)catechin before forced-aging resulted in a significant (P < 0.05) decrease in peaks 1, 10, 17, 18, 19, 20, and 21, while the addition of ferulic acid resulted in a significant (P < 0.05) increase in peaks 10, 18, 21, 23, 24, and 25 (12). It would appear from these results that the addition of the antioxidants (+)catechin and ferulic acid to beer has no impact on carbonyl formation when oxygen levels are <0.1 ppm, irrespective of the storage temperature. However, the addition of air to the package followed by heating resulted in the formation of additional carbonyl compounds. The presence of (+)catechin or ferulic acid in the beer altered the rate of formation of some of these additional carbonyl compounds. The amount of trans-2-nonenal in the beer increased dramatically during storage and the rate of formation increased with increasing temperature (Fig. 8). During forced-aging there was only a very small increase in the amount of trans-2-nonenal with increasing temperature. The addition of (+)catechin or ferulic acid had no impact on its rate of formation. The rate of formation would appear to be dependent on storage temperature and time but not on the addition of air under the experimental conditions described here. In contrast, undecanal did not increase during storage, irrespective of temperature. However, it did increase upon addition of air before heating. The presence of (+)catechin reduced its rate of formation. There were other unidentified carbonyl compounds that eluted in the same region of the chromatogram as undecanal that responded in a similar way. As these compounds are likely to be long-chain carbonyls, they would be expected to have lower flavor thresholds. In combination, these compounds may contribute to stale flavors in beer aged under exposure to air. Summarizing, it would appear that trans-2-nonenal is still formed under packaging conditions capable of delivering oxygen levels <0.1 ppm. The antioxidants (+)catechin and ferulic acid did not affect the rate of formation. This indicates that the potential to form

It is clear that during aging of beer, either for extended storage or forced-aging, there are two mechanisms for the development of carbonyl compounds. The first involves storage time and heat,

Fig. 9. Comparison of carbonyl profiles for beer stored at 40C for 72 days (A), beer forced-aged at 60C for 1 day (B), and fresh beer (C). Retention time (min) for peaks are 1: 5.70 formaldehyde; 2: 7.00 acetaldehyde; 3: 7.92 acetone; 4: 9.21; 5: 11.38; 6: 11.92; 7: 13.92; 8: 15.50 3-methyl-2-butenal; 9: 16.00 hexanal; 10: 17.00 furfural; 11: 19.07; 12: 19.97; 13: 23.50 trans-2-octenal; 14: 26.00 trans-2-nonenal; 15: 27.70 5-hydroxymethylfurfural; 16: 28.42; 17: 28.70, 18: 28.99; 19: 29.34 undecanal; 20: 29.61; 21: 30.99; 22: 31.88; 23: 32.95; 24: 34.25; 25: 35.94.

TABLE I Carbonyl Compounds Not Changed by Temperature or by Exposure to Aira Bottle Peak No 2 3 5 11 12 24
a b

Sealed Vial 60C 60C + Air 3.75 (0.21) 1.58 (0.08) 0.18 (0.04) 0.12 (0.04) 0.15 (0.02) 0.43 (0.03) 0C 2.53 (0.25) 1.39 (0.18) 0.15 (0.05) 0.12 (0.03) 0.25 (0.02) 0.26 (0.09) 0C + Air 2.56 (0.13) 1.41 (0.13) 0.15 (0.01) 0.13 (0.01) 0.30 (0.05) 0.32 (0.13) 60C 2.88 (0.20) 1.41 (0.02) 0.17 (0.02) 0.14 (0.03) 0.21 (0.02) 0.42 (0.05) 60C + Air 3.25 (0.40) 1.35 (0.08) 0.17 (0.03) 0.10 (0.02) 0.23 (0.09) 0.44 (0.19)

Peak RTb (min) 7.00 7.92 11.40 19.10 20.00 34.30

0C 3.27 (0.90) 1.36 (0.42) 0.17 (0.03) 0.13 (0.05) 0.18 (0.01) 0.22 (0.15)

0C + Air 2.73 (0.17) 1.17 (0.07) 0.16 (0.01) 0.13 (0.02) 0.18 (0.01) 0.27 (0.10)

3.05 (0.16) 1.31 (0.08) 0.18 (0.03) 0.15 (0.02) 0.13 (0.02) 0.27 (0.10)

Mean relative peak areas for each treatment. Values in parentheses are standard deviations. Retention time. TABLE II Carbonyl Compounds Not Changed by Temperature or by Exposure to Air: Summary of Two-Way Analysis of Variance P-Values Bottleb Sealed Vialc Interaction 0.054958 0.100444 0.668999 0.588658 0.337288 0.340823 Temperature 0.009126 0.733495 0.271731 0.646488 0.112417 0.089550 Air 0.233489 0.773599 0.949720 0.267733 0.310535 0.602530 Interaction 0.305506 0.572962 0.906804 0.226472 0.484858 0.758090 Peak RTa 7.00 7.92 11.40 19.10 20.00 34.30

Peak No. 2 3 5 11 12 24
a b c

Temperature 0.181213 0.196916 0.502257 0.689610 0.003521 0.089550

Air 0.783156 0.753652 0.985702 0.380648 0.224319 0.097430

Retention time. Beer stored in bottles at 0 or 60C for 22 hr. Beer transferred to vials and stored at 0 or 60C for 22 hr. (This beer was exposed to air during transfer.)

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TABLE III Carbonyl Compounds Changed by Temperature But Not by Exposure to Aira Bottle Peak No 1 4 7 8 9 10 14 15
a b

Sealed Vial 60C 60C + Air 1.87 (0.08) 0.62 (0.09) 2.22 (0.16) 0.39 (0.09) 1.62 (0.10) 1.19 (0.06) 0.04 (0.02) 2.58 (0.28) 0C 1.01 (0.14) 0.08 (0.03) 1.65 (0.27) 0.25 (0.03) 1.28 (0.13) 0.13 (0.01) 0.04 (0.03) 0.95 (0.13) 0C + Air 1.09 (0.05) 0.08 (0.00) 1.82 (0.07) 0.31 (0.00) 1.37 (0.10) 0.16 (0.03) 0.04 (0.02) 1.08 (0.14) 60C 1.73 (0.05) 0.48 (0.12) 2.26 (0.16) 0.73 (0.07) 1.61 (0.07) 1.18 (0.07) 0.05 (0.02) 2.11 (0.05) 60C + Air 1.80 (0.14) 0.66 (0.05) 2.33 (0.05) 0.64 (0.21) 1.63 (0.13) 1.15 (0.12) 0.04 (0.04) 2.25 (0.30)

Peak RTb (min) 5.70 9.21 13.90 15.50 16.00 17.00 26.00 27.70

0C 1.22 (0.52) 0.07 (0.02) 1.64 (0.40) 0.16 (0.09) 1.12 (0.26) 0.08 (0.04) 0.01 (0.02) 0.52 (0.29)

0C + Air 1.06 (0.08) 0.06 (0.01) 1.40 (0.05) 0.10 (0.01) 1.05 (0.06) 0.07 (0.00) 0.01 (0.01) 0.54 (0.10)

1.34 (0.05) 0.53 (0.09) 2.68 (0.10) 0.20 (0.06) 1.47 (0.06) 1.16 (0.06) 0.04 (0.01) 2.52 (0.36)

Mean relative peak areas for each treatment. Values in parentheses are standard deviations. Retention time. TABLE IV Carbonyl Compounds Changed by Temperature But Not by Exposure to Air: Summary of Two-Way Analysis of Variance P-Values Bottleb Sealed Vialc Interaction 0.053107 0.199341 0.404183 0.018893 0.223388 0.389868 0.559007 0.922034 Temperature 0.000002 0.000001 0.000308 0.000201 0.001948 0.000000 0.719821 0.000003 Air 0.269768 0.044563 0.241637 0.802228 0.405415 0.936178 0.829142 0.237668 Interaction 0.918373 0.044563 0.628794 0.266588 0.559535 0.497523 0.942576 0.946255 Peak RTa 5.70 9.21 13.90 15.50 16.00 17.00 26.00 27.70

Peak No. 1 4 7 8 9 10 14 15
a b c

Temperature 0.016201 0.000001 0.000088 0.003420 0.000626 0.000000 0.016780 0.000001

Air 0.271539 0.256114 0.025207 0.137622 0.644972 0.632926 0.788719 0.807413

Retention time. Beer stored in bottles at 0 or 60C for 22 hr. Beer transferred to vials and stored at 0 or 60C for 22 hr. (This beer was exposed to air during transfer.) TABLE V Carbonyl Compounds Changed by Temperature and by Exposure to Aira Bottle Sealed Vial 60C 0.06 (0.02) 0.04 (0.01) 0.00 (0.00) 0.06 (0.07) 0.03 (0.03) 0.09 (0.03) 0.05 (0.02) 0.00 (0.00) 0.01 (0.01) 0.00 (0.00) 60C + Air 0.28 (0.04) 0.09 (0.01) 0.24 (0.06) 0.28 (0.05) 0.11 (0.04) 0.18 (0.03) 0.09 (0.03) 0.06 (0.02) 0.58 (0.15) 0.02 (0.01) 0C 0.05 (0.01) 0.03 (0.01) 0.00 (0.00) 0.07 (0.01) 0.03 (0.00) 0.07 (0.02) 0.07 (0.01) 0.00 (0.01) 0.13 (0.05) 0.01 (0.01) 0C + Air 0.06 (0.01) 0.03 (0.01) 0.00 (0.00) 0.08 (0.04) 0.04 (0.01) 0.08 (0.01) 0.07 (0.02) 0.00 (0.01) 0.60 (0.30) 0.02 (0.01) 60C 0.19 (0.01) 0.17 (0.02) 0.23 (0.02) 0.32 (0.04) 0.10 (0.01) 0.17 (0.03) 0.11 (0.02) 0.05 (0.01) 1.41 (0.09) 0.06 (0.03) 60C + Air 0.20 (0.07) 0.13 (0.03) 0.27 (0.09) 0.26 (0.04) 0.11 (0.05) 0.16 (0.04) 0.12 (0.06) 0.04 (0.01) 1.49 (0.71) 0.02 (0.00)

Peak No 6 13 16 17 18 19 20 21 22 23
a b

Peak RTb (min) 11.90 23.50 28.40 28.70 29.00 29.30 29.60 31.00 31.90 33.00

0C 0.05 (0.02) 0.02 (0.02) 0.02 (0.02) 0.03 (0.02) 0.00 (0.00) 0.03 (0.02) 0.03 (0.01) 0.00 (0.00) 0.12 (0.21) 0.00 (0.00)

0C + Air 0.05 (0.00) 0.00 (0.00) 0.03 (0.03) 0.04 (0.01) 0.01 (0.01) 0.03 (0.00) 0.04 (0.00) 0.00 (0.00) 0.02 (0.03) 0.00 (0.00)

Mean relative peak areas for each treatment. Values in parentheses are standard deviations. Retention time. TABLE VI Carbonyl Compounds Changed by Temperature and by Exposure to Air: Summary of Two-Way Analysis of Variance P-Values Bottleb Sealed Vialc Interaction 0.000050 0.001993 0.000569 0.003750 0.048403 0.016260 0.201571 0.000416 0.002305 0.002488 Temperature 0.000176 0.000002 0.000011 0.000003 0.002122 0.000181 0.062866 0.000177 0.001212 0.013053 Air 0.649636 0.175836 0.557782 0.212649 0.783942 0.811556 0.728260 0.560854 0.254227 0.064889 Interaction 0.981446 0.093288 0.557782 0.122611 0.859887 0.441532 0.920637 0.457804 0.398513 0.036220 Peak RTa 11.90 23.50 28.40 28.70 29.00 29.30 29.60 31.00 31.90 33.00

Peak No. 6 13 16 17 18 19 20 21 22 23
a b c

Temperature 0.000031 0.000182 0.002241 0.000764 0.001316 0.000056 0.013845 0.000416 0.018393 0.002488

Air 0.000035 0.056169 0.000372 0.002544 0.019634 0.008852 0.054572 0.000416 0.015627 0.002488

Retention time. Beer stored in bottles at 0 or 60C for 22 hr. Beer transferred to vials and stored at 0 or 60C for 22 hr. (This beer was exposed to air during transfer.)

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Walters, M. T., Heasman, A. P., and Hughes, P. S. compounds, including undecanal. The antioxidants (+)catechin and ferulic acid were effective at reducing the rate of formation of carbonyl compounds in beer with high levels of oxygen but not in beer stored with low levels of oxygen. The addition of these antioxidants to beer before packaging is likely to have a benefit in terms of flavor stability only if the beer is exposed to air during packaging. If in-pack oxygen is minimized, the addition of these antioxidants to the beer before packaging is unlikely to improve flavor stability. In addition, there is some evidence that both (+)catechin and ferulic acid can contribute negative attributes to the beer. The presence of (+)catechin in beer has been linked to colloidal instability, whereas the degradation product of ferulic acid (4-vinylguaiacol) can contribute strong flavor and aroma to the beer that could be considered detrimental.
ACKNOWLEDGMENTS We would like to thank Charlie Bamforth, Robert Muller, and Andrew Price for their critical review of this article. We would also like to thank Louise Bolshaw for her technical skills in the analysis of the hop iso-acids. The Director-General of BRF International is thanked for permission to publish this article. LITERATURE CITED 1. Asano, K., Ohtsu, K., Shinagawa, K., and Hashimoto, N. Affinity of proanthocyanidins and their oxidation products for haze-forming proteins of beer and the formation of chill haze. Rep. Res. Lab. Kirin Brew. Co. 29:1139-1146, 1984. 2. Dadic, M., Van Gheluwe, J. E. A., and Valyi, Z. Phenolic taste in beer. Wallerstein Labs. Commun. 34:5-14, 1971. 3. Dadic, M., Van Gheluwe, J. E. A., and Valyi, Z. Formation of volatile carbonyls through the action of phenolics. MBAA Tech. Q. 11:164-165, 1974. 4. Dalgliesh, C. E. Flavour stability. Proc. Congr. Eur. Brew. Conv. 9:623-659, 1977. 5. Graf, E. Antioxidant potential of ferulic acid. Free Rad. Biol. Med. 13:435-448, 1992. 6. Grnqvist, A., Siirl, J., Virtanen, H., Home, S., and Pajun, E. Carbonyl compounds during beer production and in beer. Proc. Congr. Eur. Brew. Conv. 24:421-428, 1993. 7. Hughes, P. S. Preparative regime for the purification of bitter acids derived from hops (Humulus lupulus L.). J. Chromatogr. A. 731:327330, 1996. 8. Narzi, L., Miedaner, H., Graf, H., Eichorn, P., and Lustig, S. Technological approach to improve flavour stability. MBAA Tech. Q. 30:48-53, 1993. 9. Scott, B. C., Butler, J., Halliwell, B., and Aruoma, O. I. Evaluation of the antioxidant actions of ferulic acid and catechins. Free Rad. Res. Comms. 19:241-253, 1993. 10. Shahidi, F., and Naczk, M. Food Phenolics, Sources, Chemistry, Effects, Applications. Technomic Publishing, Lancaster, PA, 1995. 11. Walters, M. T., Hughes, P. S., and Bamforth, C. W. The evaluation of natural antioxidants in beer and its raw materials. Proc. Conv. IOB, Asia Pacific Section, Singapore. 24:103-109, 1996. 12. Walters, M. T., Heasman A. P., and Hughes, P. S. Comparison of (+)catechin and ferulic acid as natural antioxidants and their impact on flavor stability. Part 1: Forced-Aging. J. Am. Soc. Brew Chem. 55:83-89. 13. Walters, M. T. Natural antioxidants and flavour stability. Ferment 10:111-119, 1997.

this staling compound is already developed before packaging. In beer exposed to air, other long-chain carbonyl compounds, including undecanal, are formed which could contribute to the perception of an oxidized stale flavor in beer. The addition of (+) catechin to beer reduced the rate of formation of some of these oxygen-induced carbonyl compounds. Effect of Heat on Flavor Markers Storage at 25 and 0C greatly reduced the magnitude of chemical changes in beer over the five-month period compared with beer stored at 40C. In some countries, temperatures well in excess of 25C are commonplace, and beer stored for extended periods unrefrigerated could undergo the changes measured in the 40C samples. Only small changes in concentration of marker compounds were observed for temperatures between 0 and 25C. A significantly larger change in concentration was observed for temperatures between 25 and 40C. This was clearly evident for HPLC peaks 8.10 (Fig. 3), 6.03 (Fig. 4), the cis- and trans-iso-acids (Figs. 5 and 6), and the carbonyl compounds 5-hydroxymethylfurfural and trans-2-nonenal (Fig. 8). The HPLC peaks 6.59 and 7.15, and to a lesser extent furfural (Fig. 8), showed a less dramatic increase in concentration between 25 and 40C than was observed between 0 and 25C. This effect was investigated further by storing beer at different temperatures for 24 hr under forcing conditions. Under these conditions, furfural, 5-hydroxymethylfurfural, and HPLC peak 8.10 did not increase significantly with storage between 0 and 40C but showed a marked increase once storage temperatures were between 40 and 60C. The length of storage at 0C before heating had no impact on the rate of formation of the compounds with heating. This indicates that there were no changes in the beer stored at 0C for 97 days that made the beer more susceptible to changes caused by increased temperature. In countries where ambient temperatures are >30C for long periods, there is greater potential for flavor instability if beer is stored unrefrigerated either during transport or after purchase. CONCLUSIONS A large number of chemical changes occur in beer during storage, some of which are likely to affect flavor or colloidal stability. These changes include an increase in some carbonyl compounds, decrease in cis- and trans-iso--acids, and changes in UV-active compounds separated by RP-HPLC. The antioxidants (+)catechin and ferulic acid alter the rate of change of the cis-iso--acids and some UV-active compounds separated by RP-HPLC. Heat greatly affects these changes, especially at >25C. The antioxidants (+)catechin and ferulic acid have no effect on the rate of formation of carbonyl compounds that increase during storage, especially trans-2-nonenal, furfural, and 5-hydroxymethylfurfural. The carbonyl profile during storage is different from that obtained during forced-aging in the presence of air, and additional carbonyl compounds, including undecanal, are formed. These results are consistent with two mechanisms of carbonyl formation during storage of beer. The first occurs with low levels of oxygen and involves, in part, trans-2-nonenal formation. This focuses attention further upstream on the stages where trans-2nonenal is formed. The second mechanism occurs with addition of oxygen, which results in the formation of additional carbonyl

[Received January 2, 1997. Accepted May 2, 1997.]