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Total hordatine content in different types of beers

2016; Wiley; Volume: 122; Issue: 2 Linguagem: Inglês

10.1002/jib.311

2050-0416

Juha‐Matti Pihlava, Tuula Kurtelius, Timo Hurme,

Phytochemicals and Antioxidant Activities

Journal of the Institute of BrewingVolume 122, Issue 2 p. 212-217 Research articleFree Access Total hordatine content in different types of beers Juha-Matti Pihlava, Corresponding Author Juha-Matti Pihlava Natural Resources Institute Finland, 31600 Jokioinen, FinlandCorrespondence to: J.-M. Pihlava, Natural Resources Institute Finland, 31600 Jokioinen, Finland. E-mail: juha-matti.pihlava@luke.fiSearch for more papers by this authorTuula Kurtelius, Tuula Kurtelius Natural Resources Institute Finland, 31600 Jokioinen, FinlandSearch for more papers by this authorTimo Hurme, Timo Hurme Natural Resources Institute Finland, 31600 Jokioinen, FinlandSearch for more papers by this author Juha-Matti Pihlava, Corresponding Author Juha-Matti Pihlava Natural Resources Institute Finland, 31600 Jokioinen, FinlandCorrespondence to: J.-M. Pihlava, Natural Resources Institute Finland, 31600 Jokioinen, Finland. E-mail: juha-matti.pihlava@luke.fiSearch for more papers by this authorTuula Kurtelius, Tuula Kurtelius Natural Resources Institute Finland, 31600 Jokioinen, FinlandSearch for more papers by this authorTimo Hurme, Timo Hurme Natural Resources Institute Finland, 31600 Jokioinen, FinlandSearch for more papers by this author First published: 06 April 2016 https://doi.org/10.1002/jib.311Citations: 10AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Hordatines are phenolic secondary metabolites typical of barley. Hordatines withstand at least moderate processing, and thus they are also found in barley malts and beer. So far, no published data on the hordatine content has been available in beers or different styles of beer. The aim of this study was to produce information on the total hordatine content in beers and statistically compare the hordatine content of different beer types. In the current study, hordatines were analysed in 208 beers by high-performance liquid chromatography equipped with a diode array detector (HPLC-DAD). The average total hordatine content of all beer samples was 5.6 ± 3.1 mg L−1 as p-coumaric acid equivalents (PCAE), with a minimum values 0 to a maximum value 18.7 mg L−1 PCAE. The total hordatine content correlated positively to the alcohol content in lagers, ales, stouts and porters, but not in wheat beers. There was no statistically significant difference in hordatine content in different types of beer, excluding the non-alcoholic group of beers. It is noteworthy that non-alcoholic beers also contained hordatines. More research would be needed to understand how parameters, such as mashing, should be chosen in order to achieve maximum recovery of hordatines in wort and beer. Introduction Beer contains various phenolic compounds, such as phenolic acids and their conjugates, flavanols, flavonols, flavones, prenylflavonoids and hordatines 1-7, and it can be considered to be a relatively good source of phenolic compounds 8. Recently, phenolic compounds in beer have been shown to have positive effects in men with a high cardiovascular risk, by reducing leukocyte adhesion molecules and inflammatory biomarkers 9 and by increasing circulating endothelial progenitor cells 10. One of the less studied groups of phenolic compounds in beer are hordatines, which are phenolamides typical of barley (Hordeum vulgare), although small amounts can also be found in special wheats 11. Hordatines and their hydroxycinnamoylagmatine precursors in barley have been of interest since the 1960s because of their antifungal activities against plant pathogens 12-15. Hordatines are formed by the dimerization of hydroxycinnamic acid agmatines, such as p-coumaroylagmatine and feruloylagmatine, so that hordatine A is a dimer of p-coumaroylagmatine, hordatine B is a dimer of feruloylagmatine and p-coumaroylagmatine, and hordatine C is a dimer of feruloylagmatine 16-18. These three basic hordatine structures can be present as mono- or dihydroxylated forms with the hydroxyl-group located in the agmatine residues 6, 18, 19. Other hordatines are hordatine D, which includes sinapoylagmatine residue 18 and N6-methylated hordatines 6. Most of the hordatines have been found as glucosides, typically with one or two hexoside units 6, 18, 19, but in beer, hordatines even up to nine hexoside units have been found 6. Examples of the chemical structures of hordatines are presented in Fig. 1. Figure 1Open in figure viewerPowerPoint Examples of the chemical structures of hordatines. Hordatine A, R1 = R2 = H; hordatine B, R1 = OCH3, R2 = H; and hordatine C, R1 = R2 = OCH3. Of the minor compounds, monohydroxylated hordatines A1–C1 have a hydroxyl group in position X1 (or in X2) and dihydroxylated hordatines A2–C2 have hydroxyl groups in positions X1 and X2. N6-methylation can be present in position X3. Glycosylation of hordatines occurs via the hydroxyl group pointed out by the grey arrow. Naming of the hydroxylated hordatines is according to Pihlava (6). According to Kohyama and Ono 20, the concentration of hordatine A glucoside in unmalted barley varies from 103 to 254 nmol g−1 dry weight (i.e. 73–181 mg kg−1 d.w. or as hordatine A aglycon 57–140 mg kg−1 d.w.). Based on the pearling tests, hordatine A glucoside appeared to be localized in the aleurone layer of the grain 20. Based on the research by Gorzolka et al. 18, the exact localization of hordatines in barley grain would be in the outer tissues and in the interspace between the husk and the aleurone layer. In the germinated grain, hordatines are found in roots and shoots. However, there were differences in the distribution of hordatine A, B and C and their mono- and dihexoside conjugates; for instance, hordatine A was highest in the shoots, while hordatine B was the dominant form in the roots. In addition, hordatine dihexosides were concentrated in the roots, but monohexosides of hordatine A and B were more evenly distributed in the shoots and roots 18. Kageyama et al. 21 reported that certain hordatines are astringent compounds. They speculated that they might make a contribution to the aftertaste of beers. The astringent aftertaste in beer can be reduced by treating the malt by subcritical water, which will hydrolyse most of the hordatines located mainly in the acrospires 22. Originally, hordatines were found in beer while investigating compounds that were found to stimulate gastrointestinal motility by binding to the muscarinic M3 receptors, which are present in the smooth muscles of the upper gastrointestinal tract. The compounds responsible were identified as cis- and trans- forms of hordatine A 23-25. The objective of this study was to investigate the variation of the total hordatine content in various beers, and to evaluate whether beer type or style has an effect on the total hordatine content. Hordatines were analysed by high-performance liquid chromatography with a diode array detector (HPLC-DAD). To our knowledge, this is the first study, even at a semi-quantitative level, of the total hordatine contents in various beers and beer types. Materials and methods Chemicals and reagents p-Coumaric acid was purchased from Sigma-Aldrich (St Louis, MO, USA). Samples Beers (n = 208) were purchased from local grocery stores or from the national alcoholic beverage retailing monopoly of Finland (Alko Inc). One of the ales was obtained from a brew pub (Plevna, Tampere, Finland). Most of the beers were lagers (LA) (n = 114), ales (ALE) (n = 38) or wheat beers (WB) (n = 33). Minor groups were stouts and porters (SP) (n = 8). A miscellaneous group (MISC) (n = 7) included barley wines, Trappist beers and an indigenous Finnish beer sahti, although these could also be considered to be ales. A group of non-alcoholic (NA) beers also included low alcoholic 0.5% ABV (alcohol by volume) beers, and of these 13 NA beers, five were included in the WB group. Some beers in the ALE group also contained wheat as an ingredient, but as they were not referred to as wheat beers by the manufacturer, they were considered to be ales. Most of the samples were from the year 2011 (n = 173), but 35 samples were also from the preliminary experiment in 2009. In the beer samples studied, ABV varied from 0 to 17.7%. Data on the ABV and the country of origin are listed as Supporting Information in Table S1. Sample preparation A sample aliquot was taken from the bottled or canned beer to a plastic centrifuge tube and stored in a freezer. Prior to analysis, the samples were thawed at room temperature. For the liquid chromatographic analysis, samples were filtered through 0.45 µm PTFE membrane filters (Pall Corporation, Port Washington, NY, USA) into autosampler vials. HPLC-DAD analysis of hordatines Hordatines were identified and quantified using an Agilent 1100 Series high-performance liquid chromatograph (HPLC) equipped with a diode array detector (DAD) (Agilent, Waldbronn, Germany). The HPLC pumps, autosampler, column oven and diode array system were monitored and controlled using the ChemStation computer program. The analytical column was Nova Pak C18 (150 × 3.9 mm i.d., 4 um, Waters, Milford, MA, USA). The mobile phase was a gradient of 0.05 m phosphate buffer at pH 2.4 (A) and methanol (B) at 0.9 mL min−1. The eluent gradient was 5–60% B in 50 min, followed by 60–90% B in 6 min. For identification purposes, UV/vis spectra were recorded at 190–600 nm. Injection volume was 25 μL. Triplicate injections of each beer were made, except in the preliminary study, where the results were from a single injection. A specific lager beer was used throughout the HPLC analysis as a quality control sample. Hordatines were detected at a wavelength of 280 nm. Because of the lack of a commercial chemical standard, quantitation of hordatines was carried out using the calibration curve of p-coumaric acid and the results were expressed as p-coumaric acid equivalents (PCAE). Statistical analysis SAS version 9.4 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analyses. The total hordatine contents in individual beers were analyzed using an unequal slopes analysis-of-covariance model, where the means of the total hordatine content of different beer types were compared adjusting for the ABV. For easier evaluation, the total hordatine concentrations were also divided by the ABV of the beers, i.e. the results were ‘normalized’. The normalized total hordatine contents of individual beers were analysed using a one-way analysis of variance model, where the mean normalized total hordatine contents of different beer types were compared. Square root transformation was applied to the response variable in order to meet the model assumptions. Also, two individual beers had large outlying values of total hordatine content, so the analyses were performed both with and without these specific beers. The statistical models were fitted using the GLM procedures of SAS 9.4, and the model assumptions were checked using appropriate graphs. Results and discussion Hordatines in beer or malt have been subject of study in only a few cases 6, 18, 19, 21, 22 and so, at the moment, knowledge of dietary hordatines can be considered to be very limited. For example, the dietary intake or the metabolism of hordatines in humans is not known. Also, possible health effects of hordatines, despite some physiological effects 23-25, are yet to be discovered. From the analytical point of view, the lack of a proper commercial reference compound is the major hindrance for the precise determination of hordatines in diets. At least the aglycons of hordatine A, B and C would be required in order to obtain quantitative results. In this study, p-coumaric acid was used as a reference compound, since it is commercially readily available, and it is one of the structural building blocks of hordatines. One to six peaks showing typical UV spectra of hordatines were present in the chromatograms of the beer samples. An example of chromatogram and UV-spectra of hordatines are given in Fig. 2. The UV spectra were similar to the ones presented by von Röpenack et al. 14. Based on an earlier study 6, it was assumed that the major peaks would be hordatines A, B and C and their hexosides. According to our unpublished preliminary work on isolated hordatine mixtures, the conversion factor of results obtained with the calibration curve of p-coumaric acid to hordatine A or B would be roughly 5. Figure 2Open in figure viewerPowerPoint Example of the chromatogram of ale beer ID 68 at 280 nm and UV spectra of hordatines 1–6 in ale ID 68. The results of the total hordatine contents of individual beers are listed as Supporting Information in Table S1. The average total hordatine content of all beers analysed (n = 208) was 5.6 ± 3.1 mg L−1 PCAE. In different styles of beer, the average total hordatine content was as follows: ALE, 4.6 ± 3.5 mg L−1 PCAE; LA, 5.9 ± 2.2 mg L−1 PCAE; MISC, 10.9 ± 5.5 mg L−1 PCAE; SP, 6.5 ± 3.1 mg L−1 PCAE; WB, 3.2 ± 2.3 mg L−1 PCAE; and NA, 5.2 ± 2.9 mg L−1 PCAE. Hordatines were found in all beer samples except in ales ID 29, 145 and 152 and in wheat beer ID 136 (limit of detection 0.1 mg L−1 PCAE). The highest hordatine contents, 18.7 and 17.7 mg L−1 PCAE, were found in ale ID 68 (6.6 % ABV) and barley wine ID 134 (10.2% ABV) respectively. There was no statistically significant difference in the average concentrations of hordatines between different types of beers, when the NA beer group was excluded. The results of the total hordatine content, in relation to the ABVs of all beers except the NA, are given in Fig. 3 and as a box-plot presentation in Fig. 4. The relationship of ABV to the total hordatine content was different for different beer types, which was taken into account in the statistical analysis by allowing unequal slopes for different beer types 26. Owing to the unequal slopes, the pairwise comparisons between beer types yielded different results depending on the ABV level. The NA beers were excluded from this statistical analysis. Also, two individual beers (ale ID 68 and wheat beer ID 147) had a large outlying value for total hordatine, so the analyses were performed both with and without these values in order to compare the results. The total hordatine contents of ALE, LA, SP and MISC correlated positively to the ABV. This was expected with the all malt beers, since in order to obtain higher ABV beer, more malt is needed to provide a sufficient amount of fermentable sugars. Also, in stronger beers the phenolic compounds extracted from the malts are in a more concentrated form, i.e. less diluted with water. It is also expected that, when substituting barley malts with rice, corn, barley starch or other sources of fermentable sugars, lower hordatine content in the beer would result, although this was not particularly studied in this work. In WB, the correlation with the ABV was not clear and was different compared with other beer groups, which was most likely due to the different amounts of barley malts used in the production of these beers. Figure 3Open in figure viewerPowerPoint Correlation of total hordatine content [mg L-1 p-coumaric acid equivalents (PCAE)] in various styles of beer with ABV (alcohol by volume). Non-alcoholic beers are not included in the figure. Abbreviations: ALE, ale-type, top-fermented beers; LA, lager type, bottom-fermented beers; MISC, miscellaneous beers; SP, stout and porter beers; and WB, wheat beers. Figure 4Open in figure viewerPowerPoint Box-plot presentation of total hordatine content [mg L−1 p-coumaric acid equivalents (PCAE)] in all styles of beer. Abbreviations as in Fig. 3 and in addition NA, non-alcoholic beers. Results were evaluated statistically at ABV 4.5, 6.0 and 7.5%. At ABV 4.5% only the comparison of LA vs WB was statistically significant (p = 0.005). At ABV 6.0 and 7.5%, WB differed significantly (p < 0.05) from all other beer styles. When the two highest values, considered as outliers, were excluded from the ales (beer ID 68) and wheat beers (beer ID 147), comparison of ALE vs WB and MISC vs WB at ABV 4.5% came close to the significant value, p = 0.07 and p = 0.06 respectively. In addition, at ABV 6.0 and 7.5%, comparisons of ALE vs LA (p = 0.01 and p = 0.06 respectively) and ALE vs MISC (p = 0.07 and p = 0.02 respectively) were also significant or close to significant. Additional information on the statistical analysis is provided as Supporting Information (Figs S1–S5 and in Tables S2–S5). By normalization of the total hordatine content by ABV, a simpler way to compare the results was expected. The normalized average total hordatine content of all beers analysed excluding NA (n = 195) was 1.2 ± 0.5 mg L−1 PCAE ABV−1 (Supporting Information, Table S1). The normalized average total hordatine content in ALE was 1.0 ± 0.8 mg L−1 PCAE ABV−1, in LA 1.3 ± 0.4 mg L−1 PCAE, in MISC was 1.4 ± 0.4 mg L−1 PCAE ABV−1, in SP was 1.2 ± 0.5 mg L−1 PCAE ABV−1, and in WB (excluding the non-alcoholic ones) was 0.7 ± 0.5 mg L−1 PCAE ABV−1. The highest normalized hordatine contents, 2.8 and 2.7 mg L−1 PCAE ABV−1, were found in ale ID 68 (6.6% ABV) and wheat beer ID 147 (4.4% ABV), respectively. Normalized total hordatine contents are shown in Fig. 5 as a box-plot presentation. No statistically significant differences were found between different beer types or styles as regards normalized hordatine contents, when NA beers were excluded. While lower normalized hordatine content was found for instance in lager beer ID 18, in which about one-third of the barley was replaced by rice according to the information provided by the manufacturer, the hordatine content was still 1.0 mg L−1 PCAE ABV−1 and about the same as in many all-barley lagers. This could indicate variations in the hordatine content of barley malts owing to the cultivar differences 20 or different extraction recoveries of hordatines owing to differences in mashing process variables. However, this would also require more in-depth research with the knowledge of the raw materials and the details of processing. Figure 5Open in figure viewerPowerPoint Box-plot presentation of ABV normalized total hordatine content (mg L−1 PCAE ABV−1) in all styles of beer. Abbreviations as in Fig. 3. In the case of non-alcoholic beers (NA), normalization was not applied. Variation of total hordatine content in non-alcoholic beers was quite high, namely from 0.6 to 8.1 mg L−1 PCAE and in NA wheat beers from 1.6 to 5.7 mg L−1 PCAE (Figs. 4 and 5 and Supporting Information, Table S1). Also in this case, more research would be needed in order to understand how higher hordatine concentrations in non-alcoholic beers could be achieved. This could also offer exciting new possibilities for process and product development in the brewing industry. Calculating the theoretical amount of hordatine A in beer using the results given by Kohyama and Ono 20 and assuming that (a) hordatine A contents in grain and malt are the same as that of aglycon (57–140 mg kg−1 d.w.), (b) 17 kg of malt is used for brewing 100 L of beer, and (c) that the hordatine A is transferred completely to wort, the concentration of hordatine A would be 9.6–23.8 mg L−1. Although this is a very crude estimation, it at least gives some prediction of the expected amounts of hordatine at this point when no other data is available on hordatine concentrations in beers. This estimation of concentration is in line with the results presented in this study, by taking into account that, in addition to hordatine A, hordatines B and C are also present in beer and the response ratio of hordatines would be roughly 5, based on our preliminary results of isolated hordatine mixtures (data not shown). Amount-wise this would make hordatines one of the biggest groups of phenolic compounds in beer. For comparison the sum of phenolic acids, including free and bound forms, in seven beer types varied from 11.1 to 29.1 mg L−1 and the sum of free phenolic acids from 3.18 to 5.49 mg L−1 5. According to Kageyama et al. 22, concentrations of hordatines were almost the same for pale and amber malts. Based on the results of the current study of stouts and porter beers, it is not possible to draw a direct conclusion as to whether hordatines can also withstand the harsher kilning conditions used for the preparation of brown or chocolate malt. This is because the special malts form only a small part of all the barley malts used in brewing these types of beers. However, it can be concluded that hordatines are chemically at least relatively stable compounds, because the longer storage times required in the making of lager beers clearly did not result in smaller values compared with ale beers. According to our unpublished preliminary work, hordatines will also be found in considerable amounts in brewer's spent grain (data not shown). Further investigations would be required to study the effect of variables, such as the time and temperature of the mashing process, in order to reach the maximal amount of hordatines, and other phenolic compounds, in wort and beer. Similar studies have already been made mainly concerning ferulic acid, for example by Inns et al. 27 and Vanbeneden et al. 28, 29, not only because of ferulic acid's antioxidant activity but also because it is a potential flavour precursors in beers. According to Kageyama et al. 22 hordatine glycosides were rapidly and easily extracted into mash and neither the pH (4.5–5.8) of the mash nor the particle size of the malt had any effect on the concentration of hordatines. A very important question is of course the following. Would the higher concentrations of hordatines have negative attributions to the flavour of beer or other beer-like beverage? 21, 22. The main conclusions of this study are that the variation of hordatine content in beers is large, that the total hordatine content correlates positively to the ABV of beers, and that the total hordatine contents of beer types do not differ statistically significantly from each other, excluding wheat beers. More studies are needed to better understand the role and contribution of hordatines, for instance in regard to taste and possible health effects, as part of the phenolic compounds present in beer. Supporting information Supporting information can be found in the online version of this article. Acknowledgements This study was partly funded by Olvi-foundation grants. The work was conducted at MTT Agrifood Research Finland, Biotechnology and Food Research, FI-31600 Jokioinen. Dr Pirjo Mattila and Veli Hietaniemi are thanked for their valuable comments regarding the manuscript. Supporting Information Figure S3. Correlation of total hordatine content to ABV in lager beers (LA). Figure S4. Correlation of total hordatine content to ABV in stout and porter beers (SP). Figure S5. Correlation of total hordatine content to ABV in wheat beers (WB). Table S2. Statistical significances from the analysis of covariance model for total hordatine content. Table S3. Estimated means for different beer types at ABV=4.5 based on the analysis of covariance model for total hordatine content. Table S4. Estimated means for different beer types at ABV=6 based on the analysis of covariance model for total hordatine content. Table S5. Estimated means for different beer types at ABV=7.5 based on the analysis of covariance model for total hordatine content Filename Description jib311-sup-0001-Material.xlsxapplication/unknown, 24 KB Supporting info item jib311-sup-0002-Supplemenatary.docxWord 2007 document , 424.5 KB Supporting info item Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. References 1Callemien, D., and Collin, S. (2010) Structure, organoleptic properties, quantification methods, and stability of phenolic compounds in beer, Food Rev. 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