Disclaimer: Please note that I wrote this in 2012, and the field has advanced considerably since then, so information might not be up-to-date!

This is part III of the mini-essay on hop science and factors influencing hop flavor and aroma in beer. The previous part (II – Hop Oil Composition) can be found here. The topic for this post will be bitterness and how the major components of hop resin contributes to the flavor and bitterness of beer.

One of the primary uses (if not the most important use) of hops in beer brewing, is to contribute bitterness to the flavor. Hops have been used for centuries as the prime bittering agent in beer, but hops have not been the sole or original bittering agent for beer, as a variety of different herbs were used far before the use of hops became popularized. Bitterness is usually thought of as an acquired taste, i.e. something you get used to and learn to like, and bitterness perception also varies from person to person, as some can be more sensitive to it compared to others. It is also usually agreed upon that humans have through evolution learned to become alert to bitter food, as it may be a sign of poison or a possible dietary danger, and hence very bitter beers can usually be very off-putting to a person that is not used to the flavor. Bitterness research is therefore complex, but thankfully a lot of knowledge has been gained in the area.

The general structure of an alpha-acid. For humulone, R=CH2CH(CH3)2, for cohumulone, R=CH(CH3)2, and for adhumulone, R=CH(CH3)CH2CH3.

The major contributors to hop bitterness in beer are isomerized alpha-acids, however it has been found that these are not the only compounds that contribute to the bitterness (others include products derived from beta acids and polyphenols). The iso-alpha-acids are derived from alpha-acids present in the lupulin glands of the hop flower cones, and these alpha-acids are then isomerized primarily during the wort boil into iso-alpha-acids. Dried hops typically contain around 2-20% alpha-acids by weight. The three major alpha-acids present in hop resin are humulone, cohumulone and adhumulone, while two minor alpha-acids, prehumulone and posthumulone, have also been identified. These alpha-acids are all weak acids (a pKa of around 5.5), structurally related (they differ slightly via their side-chain), and are poorly soluble in acidic aqueous solutions (approximately 50 ppm for humulone in 25° C water at pH 5 and 10 ppm for humulone in 25° C water at pH 4, while a limit of 14 ppm for alpha-acids in a lager beer has also been reported). Non-isomerized alpha-acids are usually regarded as having only a slight bitter flavor, which can be tested by tasting raw hop cones or pellets, but the bitter flavor of the non-isomerized alpha-acids is not nearly as intense as the flavor of their isomerized counterparts (which is nine times stronger). The flavor threshold is also close to the maximum solubility of the alpha-acids in beer (Intelmann et al. (2009) recorded flavor thresholds of 8 ppm, 6 ppm, and 8 ppm for humulone, cohumulone and adhumulone respectively in water by a trained sensory panel, while the half-maximum bitterness intensity concentration for humulone was over 180 ppm), meaning they don’t contribute to any of the bitterness in beer.

The isomerization reaction of humulone to iso-humulone.

Thermal isomerization of the alpha-acids to iso-alpha-acids occurs via an acyloin-type ring contraction, and the reaction rate is increased with increasing temperatures. Isomerization still occurs at pre-boil temperatures, but the reaction rate at 90° C is approximately half of the reaction rate at 100° C. Isomerization forms two epimers, a trans– and a cis-iso-alpha-acid (forming in a ratio of around 1:3 in wort), meaning 6 different iso-alpha-acids are formed from the 3 major alpha-acids present in hop resin. The trans-isomers are both less bitter in flavor and less stable (i.e. more prone to oxidation) than the cis-isomers, meaning the cis-isomers are usually more desired. Foam-stabilizing properties of the cis-isomer are though slightly weaker compared to the trans-isomer. The iso-alpha-acids have pKa values around 3.0 and have much higher solubility in wort than the non-isomerized alpha-acids (predicted solubility of trans-isohumulone in water 72.5 ppm). This, along with the fact that the flavor threshold of iso-alpha-acids in water is around 6 ppm, explains why the iso-alpha-acids contribute for the majority of the bitterness in beer. The total iso-alpha-acid concentration can reach over 100 ppm in more bitter beers. The limiting factors include the poor solubility of the alpha-acids, the reaction rate of the isomerization, and the reaction rate of the degradation of iso-alpha-acids. Prolonged boil times (over approximately two hours) do not increase the iso-alpha-acid content of the wort, since after this the rate of iso-alpha-acid degradation exceeds that of alpha-acid isomerization, and the iso-alpha-acid starts to decrease. Hence, the maximum amount of bitterness one can get from a 60-minute boil addition (independent of the size of the addition) is approximately 50 IBU (see this Basic Brewing Podcast and report). If one thrives for a higher bitterness, it must be achieved from a combination of hop additions.

Table 1 – Alpha-acid and Cohumulone content of various hop varieties
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The type of alpha-acid also presumably influences the quality of bitterness, as it is generally believed that iso-cohumulone contributes a harsher and more unpleasant bitterness compared to iso-humulone. However, recent studies seem to indicate that there isn’t any difference in bittering quality between the two alpha-acids. Because of a slightly higher polarity, cohumulone and iso-cohumulone have slightly higher solubility in wort compared to humulone and iso-humulone, meaning they are usually present in slightly higher quantities. Table 1 contains a table with the typical alpha-acid percentages and cohumulone contents of various hop varieties. Looking at the table, it becomes evident that hop varieties such as Simcoe, Horizon and Warrior are preferred, if one is after a hop variety that has a low-cohumulone, yet high alpha-acid, content.

A quartz cuvette used for IBU assays. (Source)

Beer bitterness is usually measured in International Bitterness Units (IBU), or the almost identical European Bitterness Units (EBU), and it is a measure of the absorbance of a solvent extracted sample of beer at 275 nm. The absorbance is of course dependent on the amount of isomerized alpha-acids present in the sample. 1 IBU approximately converts into to 0.7 ppm iso-alpha-acid, though this depends on the presence of other bittering compouds. The IBU assay is carried out by first acidifying a sample of beer with HCl (to make the bittering acids more hydrophobic), then adding twice the volume of 2,2,4-trimethylpentane (also known as iso-octane). The mixture is then shaken thoroughly for 15 minutes, to allow as much of the (now hydrophobic) acids to transfer to the organic phase. The absorbance of the trimethylpentane phase (organic) is then measured at 275 nm in an ultraviolet spectrophotometer (against a blank). The measured absorbance is then multiplied by 50 to convert into Bitterness Units. Since the absorbance is affected by oxidation products and beta acids as well, it doesn’t give a definite picture of the amount of isomerized alpha-acids in the beer, nor the actual perceived bitterness. HPLC can instead be used to measure the amount of, and types of, isomerized alpha-acids in beer, but it requires more labor and time, as well as more costly equipment.

The general structure of an beta-acid. For lupulone, R=CH2CH(CH3)2, for colupulone, R=CH(CH3)2, and for adlupulone, R=CH(CH3)CH2CH3.

As mentioned, there are also beta-acids present in the lupulin glands of hop cones, but it is generally regarded that these don’t have a large effect on the bitterness in beer. Like the alpha-acids, there are five types typically found in hop resin (lupulone, colupulone, adlupulone, prelupulone and postlupulone) and they are structurally related weak acids (pKa of around 6). The beta-acid content of hops is usually around 1-10% by weight, and the beta-acids usually precipitate out of the wort and beer. Some of the beta-acids can though undergo transformations into bitter-tasting products such as cohulupone, hulupinic acid, nortricyclocolupone, two tricyclocolupone epimers, two dehydrotricyclocolupone epimers, two hydroxytricyclocolupone epimers, and two hydroperoxytricyclocolupone epimers. These transformation products have flavour thresholds only slightly above those of iso-alpha-acids, meaning they most likely in some way contribute to the bitterness of beer. Another series of compounds most likely contributing to the bitterness of beer are polyphenols, which are naturally found in hops, as well as malt. Polyphenols can lend a harsh and astringent flavour to the beer in large concentrations. These polyphenols can transfer to the beer through hop debris or dry hopping (polyphenols are regarded as the cause of dry hop haze), and hence even though dry hopping doesn’t contribute significantly to the amount of isomerized alpha-acids, they do contribute slightly to the bitterness of the beer through the introduction of polyphenols.

As mentioned above, cis-iso-alpha-acids are usually more stable than their trans-counterparts, meaning the ratio between them in beer usually is a good indicator over bitterness stability. To improve the bitterness stability of the beer, various techniques can be applied. The use of pre-isomerized hop products allows for a higher cis:trans ratio, post-boil whirlpool hopping has shown to increase bitterness stability, and high mash temperatures can lead to an increased amount of coagulated protein before to wort boiling and consequently less humulone-loss in the trub, which then leads to higher hop utilisation and bitterness stability. It has also been proposed that the pH of the beer affects iso-alpha-acid oxidation, though the exact effect is unclear. Isomerized alpha-acids can also undergo reduction, to form dihydroiso-alpha-acids (RIAA), tetrahydroiso-alpha-acids (TIAA) and hexahydroiso-alpha acids (HIAA). These acids are also bitter-tasting, and have improved stability against light and also increases foam stability. In water, RIAA is about 0.67 times as bitter as iso-alpha acids, TIAA 2.03 times and HIAA 1.15 times. These reduced iso-alpha-acids have similar bittering qualities to their non-reduced counterparts, and can be added to beer e.g. post-fermentation. These reduced iso-alpha-acids are usually produced using sodium borohydride, and RIAA and TIAA are available on the market for brewers.

As can be seen, hop bitterness is also a complex subject, and the traditional views on hop boiling and bitterness are slightly flawed (e.g. very high IBU values can be achieved by adding hops only post-boil). The next part will also be the final part of the mini-essay, and it will be focusing on combining the theory from this and the previous part, and applying it to actual beer brewing. I should hopefully release it in about two weeks, together with information from a trial brew.

Sources:

• Briggs, D., Boulton, C., Brookes, P., Stevens, R., 2004. Brewing: Science and Practice. Cambridge: Woodhead Publishing.
• Caballero, I., Blanco, C., Porras, M., Iso-alpha-acids, bitterness and loss of beer quality during storage. Trends in Food Science & Technology, in press
• Intelmann, D., Batram, C., Kuhn, C., Heseleu, G., Meyerhof, W., Hofmann, T., Three TAS2R Bitter Taste Receptors Mediate the Psychophysical Responses to Bitter Compounds of Hops (Humulus lupulus L.) and Beer. Chemosensory Perception 2 (2009) 118-132
• Kappler, S., Krahl, M., Geissinger, C., Becker, T., Krottenthaler, M., Degradation of Iso-Alpha-Acids During Wort Boiling. Journal of the Institute of Brewing 116 (2010) 332-338
• Khatib, A., 2006, Studies of iso-alpha-acids: analysis, purification, and stability. Doctoral Dissertation, Leiden University.
• Kishimoto, T., 2008. Hop-Derived Odorants Contributing to the Aroma Characteristics of Beer. Doctoral Dissertation, Kyoto University.
• Malowicki, M., Hop Bitter Acid Isomerization and Degradation Kinetics in a Model Wort-Boiling System, M. Sc. Thesis, 2004, Oregon State University
• Malowicki, M., Shellhammer, T., Isomerization and Degradation Kinetics of Hop (Humulus lupulus) Acids in a Model Wort-Boiling System. Journal of Agricultural and Food Chemistry 53 (2005) 4434-4439
• Schönberger, C., Kostelecky, T., 125th Anniversary Review: The Role of hops in Brewing. Journal of the Institute of Brewing 117 (2011) 259-267
• Techakriengkrai, I., Paterson, A., Taidi, B., Piggott, J., Relationships of Sensory Bitterness in Lager Beers to Iso-Alpha-Acid Contents. Journal of the Institute of Brewing 110 (2004) 51-56
• USA Hops, 2011, Variety Manual – Hop Growers of America (link)