Abstract
Recent findings have shown that besides bitter acids, several additional hop ingredients contribute to beer bitterness. These belong to the class of polyphenols and are also part of the hard resin fraction of hops. They include the two well-known flavonols, quercetin and kaempferol, widespread in the plant kingdom. On the other hand, the so-called multifidols are more typical to hops as intermediates in the biosynthesis of alpha- and beta-acids. Both of the above mentioned flavonols and the multifidols occur mainly not in their free forms but rather bonded to sugars such as glucose. In our study, the contents of such glycosidically bound polyphenols were determined in a worldwide spectrum of different hop varieties using liquid chromatography in combination with tandem mass spectrometry. In total, more than 500 samples from crops 2020 and 2019, representing 34 different varieties, were analyzed. The detected concentrations (mg/100 g) were dependent on variety, with ranges between of 6–155 for co-multifidol glucoside, 3–60 for ad-multifidol glucoside, 13–87 for quercetin-3-O-glucoside, 5–46 for quercetin-3-O-rutinoside, and 4–35 for kaempferol-3-O-glucoside. Moreover, their fate during large scale hop extraction was investigated. It was confirmed that polyphenol glycosides were not extracted with supercritical carbon dioxide but were extracted with ethanol.
Introduction
Humulus lupulus L., also named hops, is a common plant used in the brewing process and belongs to the family Cannabaceae. The beer bittering potential of hops apart from the iso-alpha-acids can be assigned to various phenolic compounds of the hard resin fraction. Based on taste re-engineering experiments, Dresel et al. could show that the additive contribution of iso-alpha-acids and various isolated hard resin components is truly necessary and sufficient for constructing the authentic bitter percept of beer.[Citation1] Besides prenylflavonoids (e.g., xanthohumol) and some esters of phenolic acids (e.g., trans-p-coumaric acid ethyl ester), this includes glycosidically bound polyphenols. Structural elements of the polyphenols are hydroxy-groups, and another important characteristic is their antioxidative activity.[Citation2]
Multifidols are acylpholoroglucinol derivatives, named after their discovery in Jatropha multifida L.[Citation3] They are also known for hops and were first extracted from Humulus lupulus L. by Bohr et al. in 2005. Three homologues could be isolated showing anti-inflammatory activity.[Citation4] These are 1-[(2-methylpropanoyl)phloroglucinyl]-β-D-glucopyranoside, 1-[(2-methylbutyryl)phloroglucinyl]-β-D-glucopyranoside, and 1-[(3-methylbutyryl)phloroglucinyl]-β-D-glucopyranoside, or more simply co-multifidol glucoside (Co-M-glc), ad-multifidol glucoside (Ad-M-glc), and n-multifidol glucoside (N-M-glc) (). Structural elucidation by Bohr et al. was conducted using NMR spectroscopy. The acyl side chains of multifidol glucopyranosides are identical to those of alpha-acids (co-, ad-, and n-humulone) because they are known intermediates in the biosynthesis of hop bitter acids from branched-chain amino acid precursors.[Citation5] Spreng and Hofmann also isolated all three multifidol homologues from a hop polyphenol extract and confirmed the structures via NMR spectroscopy,[Citation6] whereas Dresel et al. detected co-/ad-multifidol glucoside and n-multifidol di-glucoside but not n-multifidol glucoside.[Citation1] Multifidols were identified as bitter molecules having a human recognition threshold concentration of 5 μmol/L for co-multifidol glucoside and 10 μmol/L for ad-multifidol glucoside, whereas the human recognition threshold concentration for n-multifidol-di-glucoside is 37 μmol/L.[Citation1] A recent study by Morcol et al. describes co-multifidol glucoside in leaves of commercial and wild hop cultivars. This is the first time depicting this phloroglucinol derivative in wild Humulus neomexicanus.[Citation7]
Figure 1. Chemical structures of multifidol glucosides: co-multifidol glucoside (Co-M-glc), ad-multifidol glucoside (Ad-M-glc), n-multifidol glucoside (N-M-glc).
Hops contain further polyphenolic compounds bound to glycosides, which occur in many other plants. Among them are flavonol glycosides with quercetin or kaempferol as an aglycone. The most known are quercetin-3-O-glucoside (Q-glc), quercetin-3-O-rutinoside (Q-rut), and kaempferol-3-O-glucoside (K-glc) (), which are also called isoquercitrine, rutin, and astragaline. Besides many other polyphenolic hop substances (including co-multifidol glucoside), Kavalier et al. determined the contents of these three flavonol glycosides in two hop varieties from the USA (Willamette and Zeus) over different developmental stages.[Citation8] A principal component analysis of the world hop collection concluded that the contents of quercetin and kaempferol (and co-multifidol) glycosides in hops are variety dependent and therefore suitable for variety differentiation as performed by Kammhuber.[Citation2] Dresel et al. described quercetin-3-O-glucoside and kaempferol-3-O-glucoside as bitter molecules with low threshold concentrations.[Citation1] In a further study, Dresel et al. investigated a large spectrum of 75 different international hop varieties and various hop products on totally 117 single key-bitter tastants from hops and proved that glycosidically bound flavonols (and multifidols) occurred in all hop cultivars.[Citation9] Spreng and Hofmann showed that quercetin-3-O-glucoside and kaempferol-3-O-glucoside have a higher antioxidative activity than multifidol glucosides.[Citation6]
Figure 2. Chemical structures of flavonol glycosides: quercetin-3-O-glucoside (Q-glc), quercetin-3-O-rutinoside (Q-rut), and kaempferol-3-O-glucoside (K-glc).
Schmidt and Biendl investigated the amount of hard resin polyphenols in various Pale Ales produced with five different US hop varieties and displayed that such compounds clearly increased after dry hopping.[Citation10] This previous work performed in our laboratory included the analysis of multifidol and flavonol glycosides in beer after direct measurement by liquid chromatography—tandem mass spectrometry (LC-MS/MS). The results demonstrated that beers produced with different hop varieties showed significant differences in their pattern of glycosidically bound polyphenols. In addition, the contribution of these compounds to the overall bitter profile of beer was feasible when comparing the quantitative data with taste thresholds as published by Dresel et al.[Citation1] Now this current study profits from the developed LC-MS/MS method, which also allows for the monitoring of glycosidically bound polyphenols in hops and hop products, with a focus on multifidol and flavonol glycosides.
Experimental
Reagents
The following chemicals were obtained from commercial sources: water and methanol for LC-MS (Chemsolute®, Th. Geyer GmbH & Co. KG, Renningen, Germany); formic acid and ammonium formate (Merck, Darmstadt, Germany); quercetin-3-O-glucoside, quercetin-3-O-rutinoside, and kaempferol-3-O-glucoside (Merck, Darmstadt, Germany); internal standard dicamba (LGC Standards GmbH, Wesel, Germany). The purified standards for the compounds co-multifidol glucoside and ad-multifidol glucoside were provided by Dr. Philip Wietstock from the Technical University of Berlin after isolation according to Kunz et al.[Citation11] The correct structure of both standards was verified at Technical University of Munich (group of Prof. Herbert Riepl) using 400 MHz NMR spectroscopy.
Sampling
The investigated samples from Europe were raw hops (dried cones), pellets, and hop extracts taken during process control in our large-scale pellet and extraction plants throughout the entire campaigns 2019/2020, and 2020/2021. Each sample was representative of variety specific batches, with sizes up to 30 tons of raw hops or hop product, respectively. In total more than 500 single samples from 19 different varieties of the main European growing areas were analyzed. Most came from Germany, the others from the Czech Republic, Slovenia, and Poland.
In addition, a few pellet samples outside of Europe were supplied by Hopsteiner, USA (S. S. Steiner, Inc., Yakima). In most cases, these only represented two samples per variety coming from two crop years (2019, 2020). For three varieties, only one sample from one crop year was available. In total, 27 single samples from 12 different US varieties were investigated, two samples were from Australia, and one was from New Zealand.
Sample preparation
One gram of milled hop cones or pellets was weighed into a 100 mL screw cap bottle and 20 mL of HPLC solvent mixture (solvent A + solvent B, 50/50, v/v—see below) was added. The sample was extracted with the help of a shaking device (225 rpm) for 30 min. An aliquot of this sample was centrifuged for 15 min at 13,500 rpm and 1 mL of the supernatant was analyzed using LC-MS/MS in the negative mode, after addition of dicamba as an internal standard (c = 200 ng/mL).
An aliquot of 0.5 g of hop extract (total resin extract or carbon dioxide extract) was weighed into a centrifuge tube and 5 mL of the HPLC solvent B was added. The sample was extracted with the help of an ultrasonic bath (15 min). An aliquot of this solution was mixed with the HPLC solvent A to achieve a mixture of solvent A + solvent B of 50/50 (v/v) and then centrifuged at 13,500 rpm for 15 min. As described above for pellets and hop cones, 1 mL of the supernatant was analyzed, in the negative mode, after the addition of an internal standard (dicamba, c = 200 ng/mL).
Sample analysis with liquid chromatography—tandem mass spectrometry (LC-MS/MS)
HPLC system (Shimadzu Corporation, Kyoto, Japan) consisting of a binary pump, a degasser, an auto-sampler, and a column oven was coupled with a 3200 Q-TRAP mass spectrometer or a 5500+ Q-TRAP mass spectrometer (both SCIEX, Darmstadt, Germany) equipped with an electrospray ionization (ESI) source running in the negative ion mode. Samples were introduced by HPLC at a solvent flow of 500 μL/min, which required the use of turbo gas at a temperature of 480 °C (3200) and 350 °C (5500+). The ion spray voltage was set to −4500 V. Nitrogen was used as the collision gas. The collision energy (CE), the declustering potential (DP), and the cell exit potential (CXP) were optimized for each compound prior to analysis and were set as given in . The quantitation was conducted using the scheduled multiple reaction monitoring (MRM) mode. Data processing was performed by Analyst Software Version 1.7.1 and data integration was performed by MultiQuant Software Version 3.0.3 and OS version 1.7 (SCIEX, Darmstadt, Germany). For chromatography, an analytical 50 × 2.0 mm Synergi 4 µ Fusion-RP 80 A column (Phenomenex, Aschaffenburg, Germany) equipped with a guard column of the same type (Phenomenex, Aschaffenburg, Germany) served as the stationary phase. Ammonium formate (5 mM) containing 0.1% formic acid in water was used as solvent A and methanol with 5 mM ammonium formate and 0.1% formic acid was used as solvent B. The temperature of the column oven was set at 40 °C. The injection volume was 10 µL (3200) and 2 µL (5500+). Chromatography was performed by increasing solvent B from 20 to 100% within 8 min and holding for 2 min. Quantitation was conducted by external calibration (with dicamba as an internal standard) in a range between 100 and 10,000 ng/mL.
Table 1. Specific mass transitions and optimized parameters for the LC-MS/MS analysis of the selected glycosidically bound polyphenols quercetin-3-O-glucoside (Q-glc), quercetin-3-O-rutinoside (Q-rut), kaempferol-3-O-glucoside (K-glc), co-multifidol glucoside (Co-M-glc), ad-multifidol-glucoside (Ad-M-glc), and an internal standard (Dicamba).
The following recovery rates were determined: 80% for quercetin-3-O-glucoside, 80% for quercetin-3-O-rutinoside, 72% for kaempferol-3-O-glucoside, and 70% for the two multifidol glucosides.
The relative standard deviation was determined using a control sample that was a mixture of different hop varieties. This control sample was analyzed for several months (n = 74). The relative standard deviations (%RSD) were ≤20% depending on the compound: co-multifidol glucoside (Co-M-glc): 11%RSD, ad-multifidol glucoside (Ad-M-glc): 13%RSD, quercetin-glucoside (Q-glc): 14%RSD, quercetin-rutinoside (Q-rut): 20%RSD, and kaempferol-glucoside (K-glc): 16%RSD.
Results and discussion
Influence of hop variety, crop year and origin on co- and ad-multifidol glucoside contents
Contents of glycosidically bound polyphenols were monitored in raw hop and pellet samples representing a total of 34 different hop varieties of two crops (2019 and 2020) from large worldwide growing areas (Germany, Czech Republic, Slovenia, Poland, USA, Australia, and New Zealand).
The results for co-multifidol glucoside (Co-M-glc) and ad-multifidol glucoside (Ad-M-glc) are given as mean values in and , each sorted by the number of samples analyzed per variety. In comparison to most of the European hop varieties, so far only a small number of samples from the USA, Australia, and New Zealand have been investigated. The hop cultivars and their national codes are abbreviated according to the international list of hop varieties published by the International Hop Growers’ Convention (IHGC).[Citation12]
Table 2. Concentrations of co-multifidol glucoside (Co-M-glc) and ad-multifidol glucoside (Ad-M-glc) in hop varieties from Europe (Germany, Czech Republic, Slovenia, and Poland).
Table 3. Concentrations of co-multifidol glucoside (Co-M-glc) and ad-multifidol glucoside (Ad-M-glc) in hop varieties from USA, Australia, and New Zealand.
The minimum and maximum total amounts (∑M-gl) of the two multifidol glucosides (sum of co-multifidol glucoside and ad-multifidol glucoside) of all European varieties presented in range from 8.8 mg/100 g for German Hersbrucker (DE HEB, 2019) up to 192.8 mg/100 g for German Mandarina Bavaria (DE MBA, 2019). Both varieties are also the most extreme regarding co-multifidol glucoside alone with only 6.0 mg/100 g up to maximal 155.0 mg/100 g. The range for ad-multifidol glucoside is between 2.8 mg/100 g (DE HEB, 2019) and 60.2 mg/100 g in Hallertauer Taurus (DE HTU, 2020) from Germany. For each European variety presented in , the amount of co-multifidol glucoside is always higher as compared to ad-multifidol glucoside. The German high alpha variety Polaris (DE PLA) shows the highest value for the ratio of the two homologues (Co/Ad) both in 2020 (4.7) and 2019 (5.3), whereas Hallertauer Taurus (DE HTU), another German bitter variety, always shows the lowest (1.6 in 2020 and 1.9 in 2019).
Obviously, there is no correlation between the concentrations of multifidol glucosides and alpha-acids. For example, in 2020 the two highest amounts for the sum of the two homologues can be observed for the German aroma hop Hallertauer Tradition (DE HTR: 177.4 mg/100 g) and the German bitter hop Herkules (DE HKS: 164.6 mg/100 g) although their average alpha-acid contents are quite different (6.3% and 16.6% in crop 2020).[Citation13] On the other hand, the aroma variety Hersbrucker (DE HEB) is extremely low in both co- and ad-multifidol glucosides with the corresponding sum below 15 mg/100 g in crop 2020 and even below 10 mg/100 g in crop 2019. For all other varieties, this sum is always at least 40 mg/100 g.
shows the maximum amount of co-multifidol glucoside for US Comet (US COM) at 70.2 mg/100 g (2019) and thus less than half of the maximal concentrations above 160 mg/100 g as given in some hops from Europe. However, the extremely low contents for ad-multifidol glucoside in US Lemondrop (US LDP) of both crops (2.6 mg/100 g, and 2.0 mg/100 g) are even a bit lower than in German Hersbrucker (DE HEB). Interestingly, in both crops the three US hop varieties Sultana (US SUL), Mosaic (US MOS), and Calypso (US CPO) are showing higher amounts of ad-multifidol glucoside in comparison to co-multifidol glucoside. The other varieties presented in have ratios above 1, like all hops grown in Europe. The most extreme ratios of the two homologues detected so far in all varieties worldwide are for US Lemondrop (US LDP) with 7.0 in 2020, and 8.6 in 2019.
Again, no correlation between the alpha-acids amounts and the detected contents of multifidol glucosides can be observed for the varieties presented in . For example, the US high alpha hop Eureka (US EUE) shows one of the lowest sums of the two homologues (17.0 mg/100 g in 2020, and 17.8 mg/100 g in 2019) but in case of the US aroma hop Cascade (US CAS), with considerably less alpha, this total amount is on average a factor of three higher in both crops (50.2 mg/100 g, and 47.0 mg/100 g).
In summary, for almost all cultivars presented in the levels are clearly below the values of . However, this result still needs to be verified by the analysis of a larger number of samples outside of Europe.
The n-homologue of multifidol glucoside
For hop alpha-acids, the concentration of ad-humulone is relatively constant and lower than that of co- and n-humulone. For the acylphloroglucinol glucosides, it is known that co- and ad-multifidol glucosides are more abundant than the n-homologue.[Citation4] A standard for n-multifidol glucoside was not available for our studies. However, according to the structure elucidation of Bohr et al.,[Citation4] this substance differs only in the acyl moiety from ad-multifidol glucoside () with the same m/z of 371.0. Comparison with chromatographic data as given in the thesis of Spreng[Citation14] enabled a tentative identification of the peak at 3.5 min with the mass transition m/z 371.0 → 209.1 as n-multifidol glucoside (). For final confirmation, a standard would be necessary. To evaluate the occurrence of n-multifidol glucoside in comparison to co- and ad-multifidol glucoside depending on different hop varieties, the peak area for the mass transition m/z 371.0 → 209.1 at 3.5 min was compared with the area of ad-multifidol glucoside for the same mass transition. A selection is presented in with the three hop varieties Saaz (SAZ) from Czech Republic, Topaz (TOP) from Australia and Calypso (CPO) from the USA.
Figure 3. Extracted ion chromatogram (XIC) of co- and ad-multifidol glucoside for the total chromatographic run and the detailed extracted ion chromatogram (XIC) for ad-multifidol glucoside and the potential n-multifidol glucoside for three different hop varieties: Saaz (SAZ), Topaz (TOP), and Calypso (CPO).
shows both the extracted ion chromatogram of co- and ad-multifidol glucoside and the magnified extracted ion chromatogram of ad-multifidol glucoside and the potential n-multifidol glucoside. As can be seen, there are clear differences between the ratios of co- and ad-multifidol glucoside. For the varieties Saaz (SAZ) and Topaz (TOP), the amount of co-multifidol glucoside is much higher than ad-multifidol glucoside, whereas for Calypso (CPO) the amounts are closer to 1/1. Next to the different concentrations of co- and ad-multifidol glucoside, the three hop varieties show some differences in the amount of tentatively identified n-multifidol glucoside. Despite their similar levels of co- and ad-homologues, Saaz (SAZ) shows a much higher amount of the potential n-multifidol glucoside than Topaz (TOP). Calypso (CPO) is comparably low but has rather little concentrations of all three homologues.
As shown in these examples, in all other investigated samples the peak of the potential n-multifidol glucoside was always lower as compared to ad-multifidol glucoside. For exact quantification, a calibration standard of the n-homologue is needed.
Co-multifidol glucoside and ad-multifidol glucoside concentration ranges within the hop varieties Herkules and Perle
Because samples were collected throughout the ongoing large-scale hop processing campaigns, in the end most of them were available from the two most processed varieties grown in Europe (i.e., Herkules (DE HKS, bitter hop) and Perle (DE PER, aroma hop), both from Germany). For example, in 2020 a total of 144 Herkules samples (110 raw hops, 34 pellets) were analyzed and 55 Perle samples (32 raw hops, 23 pellets) were analyzed.
Such a large number of single samples allow for clear insights in the range of polyphenol glycosides within one variety. In addition to the mean values given in , the results for min/max values as well as the comparisons of raw hops with pellets are presented in for the crop years 2019 and 2020. For example, in 2020 the observed concentration range of co-multifidol glucoside for the hop cultivar Herkules was from 70.1 up to 185.7 mg/100 g for raw hops and from 73.3 to 153.8 mg/100 g for pellets. This demonstrates a wide but similar spread in both cases. The concentration ranges for Herkules in crop 2019 and for the variety Perle in both crops were at comparable orders of magnitude. Similar observations can be made for ad-multifidol glucoside but at lower levels.
Table 4. Co-multifidol glucoside (Co-M-glc) and ad-multifidol glucoside (Ad-M-glc) concentrations in mg/100 g as min/max and mean values for the German bitter variety Herkules (DE HKS) and the German aroma variety Perle (DE PER) in raw hops and hop pellets for the crop years 2019–2020 and literature data for crop 2011.[Citation9]
Such large varietal ranges make the evaluation of a high sample number per variety obvious. Moreover, it makes a comparison with literature data rather difficult, especially if the result represents only one sample as for example given by Dresel et al.[Citation9] In this paper, the concentration of co-multifidol glucoside for Perle in crop 2011 (74.1 mg/100 g) was reported on to be less than half as compared to Herkules (166.3 mg/100 g). However, the results based on a very large number of samples show rather similar levels for both varieties, even in two crop years. Nevertheless, the single numbers published by Dresel et al.[Citation9] are just still covered by the min/max ranges of our investigations. Similar conclusions can be drawn for the ad-multifidol glucoside contents in both varieties ().
Because of the observed high fluctuations, we would judge mean values only to be representative enough if at least 10 samples (of big commercial lots) per variety are analyzed. If the number of samples is between 3 and 9, the data only indicate a varietal trend and in the case of only 1 or 2 samples it is not more than a snapshot and requires further verification by follow-up analyses. These three categories must be considered when judging all mean values presented in this study.
As already discussed, according to , in both crops there were no significant differences of min/max and mean values between hop cones and pellets. Such a result was also true for all other investigated varieties as well as for the concentrations of the three flavonol glycosides (data not shown). Therefore, the averages of each analyzed parameter presented in the and were always calculated as the total mean from both cones and pellets per variety.
Table 5. Concentrations of quercetin-3-O-glucoside (Q-glc), quercetin-3-O-rutinoside (Q-rut), and kaempferol-3-O-glucoside (K-glc) in hop varieties from Europe (Germany, Czech Republic, Slovenia, and Poland).
Influence of hop variety, crop year and origin on flavonol glycoside contents
Three flavonol glycosides (quercetin-3-O-glucoside, quercetin-3-O-rutinoside, and kaempferol-3-O-glucoside) were chosen for evaluation as the most relevant representatives of the glycosidically bound polyphenols, in addition to the acylphloroglucinol glucosides. and summarize their concentrations for the harvest years 2019 and 2020.
Table 6. Concentrations of quercetin-3-O-glucoside (Q-glc), quercetin-3-O-rutinoside (Q-rut), and kaempferol-3-O-glucoside (K-glc) in hop varieties from USA, Australia, and New Zealand.
The amount of quercetin glucoside (Q-glc) is between 12.5 mg/100 g (DE HMG, 2020) and 86.5 mg/100 g (PL MAR, 2019). Interestingly, the German aroma hop Hersbrucker (DE HEB), with clearly the lowest contents of both multifidol glucosides (see ), is one of the varieties with the highest amounts of quercetin glucoside (55.1 mg/100 g in 2020, and 52.7 mg/100 g in 2019) as well as the two other flavonol glycosides. Quercetin rutinoside (Q-rut) was detected in the range of 7.6 mg/100 g (DE PLA, 2020) up to 32.5 mg/100 g (DE HMG, 2019), whereas kaempferol glucoside (K-glc) was between 3.7 mg/100 g (DE HMG, 2020) and 35.0 mg/100 g (DE HEB, 2019). Especially, the German bitter variety Hallertauer Magnum (DE HMG) appears to be very extreme with the highest content of quercetin rutinoside but the lowest for quercetin glucoside and kaempferol glucoside at the same time. Moreover, it is the only European variety with higher quercetin rutinoside levels as compared to both glucosides. This outstanding position could be confirmed for both crop years. For all varieties and the two crops, the concentration of quercetin glucoside is always clearly higher as compared to kaempferol glucoside (at least by a factor of approximately two in all cases). The levels of quercetin rutinoside and kaempferol glucoside are rather similar but with the only exception for German Hallertauer Magnum (DE HMG).
When dividing the sum of the two multifidol glucosides (∑M-gl, see ) by the total amount of the three flavonol glycosides (∑F-gl), this ratio (∑M-gl/∑F-gl) is always higher than 1 in most European varieties with only a few exceptions between 0.4–0.9 for the aroma varieties German Spalter Select (DE SSE), Slovenian Aurora (SI SSA), Slovenian Celeia (SI SGC), Slovenian Bobek (SI SGB), and Polish Marynka (PL MAR) in both crop years. However, German Hersbrucker (DE HEB) is the most extreme with an exceptional ratio of only 0.1.
For the hop varieties grown outside of Europe, the amount of quercetin glucoside is between 25.3 mg/100 g (US CPO, 2019) and 87.4 mg/100 g (US MOS, 2019). For quercetin rutinoside, the range is from 4.8 mg/100 g (US LDP, 2019) up to 45.9 mg/100 g (AU TOP, 2019), and for kaempferol glucoside it is from 4.3 mg/100 g (US CPO, 2019) up to 27.4 mg/100 g (AU TOP, 2019). These two varieties are the most outstanding. Whereas US Calypso (US CPO) shows the lowest concentrations for the two glucosides, Australian Topaz (AU TOP) is the highest for quercetin rutinoside and for kaempferol glucoside. Despite the low number of samples analyzed to date, the above-mentioned trend of minimal and maximal concentrations in different varieties can be observed for both crops. Moreover, like in the case of all European hops, the concentration of quercetin glucoside is always clearly higher as compared to kaempferol glucoside. However, in addition to German Hallertauer Magnum (DE HMG) there are some more varieties grown outside of Europe that are showing higher levels of quercetin rutinoside as compared to quercetin and kaempferol glucosides. This is true for the two US varieties Lotus (US LOT) and Cascade (US CAS), as well as for Topaz (AU TOP) and Galaxy (AU GXY), both grown in Australia, and finally also for Nelson Sauvin (NZ NSN) from New Zealand. Contrary to most of the European varieties, the ratios of total multifidols (from ) to total flavonol glycosides (from ) are always rather low. Only for the variety US Comet (US COM), it is above 1 (1.2, crop 2020). The minimum ratio is 0.1 for US Citra (US CIT) in both crop years, and thus as low as for German Hersbrucker (DE HEB, see ).
When adding up the amounts of all three flavonol glycosides, from all investigated varieties Polish Marynka is showing the highest sum with 142.8 mg/100 g (PL MAR, 2019), which is about 75% of the maximum sum of the two multifidol glucosides presented in and (192.8 mg/100 g, DE MBA). On the other hand, the lowest sum of all three flavonol glycosides is 42.0 mg/100 g for US Calypso (US CPO, 2019), which is much higher than the minimum sum of the two multifidol glucosides with 8.8 mg/100 g (DE HEB) from and . Thus, the concentration ranges for the two multifidols are more extreme as compared to the three flavonols.
In summary, all of the observations as discussed on basis of the various data sets presented in appear to be given for both crop years indicating varietal features. However, as already pointed out, in a strict sense this conclusion is only valid for varieties with at least ten investigated samples per crop year.
Transfer to hop extracts
Nowadays there are two extraction processes established in the hop industry. Whereas several extraction plants around the world are using carbon dioxide (mostly in supercritical and rather seldom in liquid phase), there is only one location with a large-scale hop extraction based on ethanol as a solvent. The main goal of all commercial hop extraction processes is to recover alpha-acids in yields of at least 95% from the hop starting materials. The actual composition of the resulting extracts is rather similar, with only minor differences depending on the polarity of extraction solvents. Lower polarity corresponds with more selective processing. Thus, liquid carbon dioxide mainly dissolves alpha- and beta-acids, whereas parts of unspecific soft resins and all the hard resins remain in the spent hops. The total amount of soft resins is only extracted with supercritical carbon dioxide. In addition, the more polar ethanol also dissolves parts of the hard resins. Since the positive contributions of the hard resin compounds to beer bitterness has become known,[Citation1] such a composition means a unique selling proposition. Therefore, this type of extract has been recently renamed from “ethanol extract” to “total resin extract.”[Citation15]
From the studies of Dresel et al.,[Citation1] it is already common knowledge that contrary to extraction with ethanol, all of the single components of the hard resin fraction are not soluble in supercritical carbon dioxide. To obtain deeper insights and more data, our study also included several samples of the two different extract types produced on a large scale from crop 2020. Thereby it could be verified that glycosidically bound polyphenols were only transferred to the so-called total resin extract and not to the supercritical carbon dioxide extract.
A comparison of the already presented average concentrations in raw hops/pellets (see ) with the mean levels in total resin extract is shown in for the German varieties Herkules (DE HKS), Hallertauer Tradition (DE HTR), Perle (DE PER), Hallertauer Taurus (DE HTU), Hallertauer Magnum (DE HMG), and Polaris (DE PLA). All investigated compounds could be detected in the total resin extract samples. For co-multifidol glucoside, ad-multifidol glucoside and kaempferol glucoside, the average extract levels reached up to 50% of the raw hop/pellet mean values, whereas for quercetin glucoside and quercetin rutinoside the recoveries were lower.
Figure 4. Bar graphs for the average contents of the multifidol glucosides (Co-M-glc and Ad-M-glc) and the flavonol glycosides (Q-glc, Q-rut and K-glc) in pellets and raw hops (total bar) as compared to their average amounts in total resin extract (striped) for the hop varieties Herkules (n (pellets and raw hops) = 144, n (total resin extract) = 12), Hallertauer Tradition (n (pellets and raw hops) = 47, n (total resin extract) = 3), Perle (n (pellets and raw hops) = 55, n (total resin extract) = 3), Hallertauer Taurus (n (pellets and raw hops) = 5, n (total resin extract) = 3), Hallertauer Magnum (n (pellets and raw hops) = 29, n (total resin extract) = 5) and Polaris (n (pellets and raw hops) = 7, n (total resin extract) = 3) in crop 2020.
Conclusion and outlook
LC-MS/MS technique enables the monitoring of different glycosidically bound polyphenols in several hop varieties from Europe, USA, Australia, and New Zealand. The average concentrations of two multifidol glucosides and three flavonol glycosides appear to be varietal features but with wide ranges within one variety. For reliable variety specific values, many samples are therefore needed. In addition to numerous analyses of raw hop and pellet samples, evaluations of hop extracts proved the absence of these compounds in the carbon dioxide extract and confirmed their presence in the ethanol hop extract, nowadays called total resin extract. The quantitative data of these glycosidically bound polyphenols are useful to determine their transfer from hops to beer and, in combination with taste thresholds known from literature, to evaluate their contribution to overall beer taste. Although these glycosides are minor constituents of hops and beer, they are known as relevant parts of the hard resin fraction with a positive impact on bitterness. They can make the taste more balanced in comparison to the bitter impression of only iso-alpha-acids. Further studies are planned, including additional phenolic compounds of hops as well as investigating the fate of such a broad range of substances during the brewing process.
Disclosure statement
No potential conflict of interest is reported by the authors.
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