Hop Freshness – Part 2: Hop Bitter Acids

Following on from Part 1: Hop Degradation and the Supply Chain, this post will focus on hop bitterness. I’ll start with a short primer on hop chemistry before considering how the bittering quality of hops can change with age and storage conditions.

Hop composition

Whole hops are comprised of numerous components as shown in Table 1. The exact proportions of these constituents is liable to change, depending on hop variety and growing conditions. The lupulin glands, located on the leaves of the hop cones, contain a complex mixture of substances including resins and essential oils. Most of the bittering value to brewers is contained within the hop resin component, while much of the aroma contribution stems from the essential oils.

Table 1. Average chemical composition of dried hop cones. Adapted from Almaguer et al. (2014).1

Constituent Amount (%)
Total resins 15-30
Essential Oil 0.5-3
Proteins 15
Monosaccharides 2
Polyphenols (tannins) 4
Pectins 2
Amino acids 0.1
Waxes and steroids Trace-25
Ash 8
Moisture 10
Cellulose, etc. 43

Hop resins and bitter compounds

Hop resins are commonly described in terms of non-specific fractions. The non-specific resin fractions and their average chemical compositions for fresh dried hop cones are shown in Fig. 1. The naming of the various resin fractions is based upon how they’re physically separated, for example, the total resin fraction is characterized by solubility in both cold methanol and diethyl ether whereas the total soft resin and total hard resin fractions are distinguished from each other by solubility of the soft resins in hexane. The total soft resin fraction and particularly the α-acids are ultimately responsible for providing the majority of the beer bittering contribution from hops.

Fig. 1. Classification of hop resins. Adapted from Almaguer et al. (2014).

It’s a slightly confusing classification system but it’s very much functional as opposed to ideal. If there was a better method for resin separation and extraction everyone would be delighted.

The soft resin consists of two main sub-fractions, the α-acids and β-acids, in addition to a non-specific fraction of uncharacterized soft resins. Fractions such as the α-acids and β-acids are well defined and contain a mixture of specific compounds in various proportions. On the other hand the uncharacterized soft resin fraction, as the name suggests, consists of a mixture of as yet uncharacterized compounds.

α-Acids

Fig. 2. Chemical structures of the three main α-acids or humulones (co/n/ad).

The α-acid or humulone fraction has been the subject of extensive research and is known to consist of a number of analogous compounds. It’s separated from the β-fraction by it’s ability to form an insoluble lead salt with lead acetate in methanol. The three major α-acids are humulone, cohumulone and adhumulone (Fig. 2), which are isomerized during the wort boiling process to form the more water-soluble cis and trans-iso-α-acids. It’s been shown that α-acids on their own do not contribute any perceivable bitterness to beer and combined with their low solubility makes their influence on beer flavour negligible.2 Iso-α-acids on the other hand are the major bittering compounds in beers brewed using fresh hops and hop products.3

β-Fraction

Fig. 3. Chemical structures of the three main β-acids or lupulones (co/n/ad).

The β-fraction is comprised of the β-acids or lupulones and the uncharacterized soft resins. The β-acids, like the α-acids, are a group of compounds with similar and closely related chemical structures, existing in three major analogous forms, known as lupulone, colupulone and adlupulone (Fig. 3). The β-acids have an additional 5-carbon isoprenyl- (3-methyl-2-butenyl-) group in place of the tertiary hydroxyl (OH) group of the α-acids, making them more hydrophobic and consequently even less water-soluble than α-acids. Also, unlike the α-acids, β-acids are not isomerized into a more soluble form during wort boiling. As a result the β-acids are largely removed by precipitation during brewing and have a negligible effect on beer flavour. Nonetheless there is some evidence that a number of bitter β-acid transformation products can be generated during wort boiling that are able to contribute to beer bitterness. One of these, cohulupone, is discussed in the oxidised β-acid section.

Hard resin fraction

The hard resin fraction is distinguished from the soft resins by it’s insolubility in hexane. The hard resins are generally believed to be the oxidation products of the soft resins and it’s been shown that as hops age during storage the proportion of hard resins increases while the amount of soft resins decreases.4 The study of this fraction is complicated by the need to distinguish between native hard resins, which have been detected in the initial stages of hop growth and those formed by oxidation reactions during drying and storage post-harvest. Recent work in this area has established that the hard resins do have some brewing value and that a “desirable mild bitterness” was imparted in beers brewed entirely from hard resin extracts.5 It’s been observed that as the α- and β-acid content decreases during storage the uncharacterized soft resin initially increases, then begins to decrease, followed by a steady increase in the hard resin content.6 It’s believed that the uncharacterized soft resins may be intermediate deterioration compounds of the α- and β-acids, which will eventually become hard resins, however it doesnt appear to be fully understood yet whether they have any brewing value.

Why hop resins deteriorate

As hops get older they begin to deteriorate (don’t we all) and as mentioned the α- and β-acids portion of the soft resins decreases while the size of the uncharacterized soft resin and hard resin fractions increases.7 The route by which hops are degraded is generally thought to be through direct contact with atmospheric oxygen, resulting in the loss of the α- and β-acids through oxidation reactions. The mechanism is still not fully understood but work in this area is on-going.

Interestingly there’s some evidence to suggest that the enzyme α-acid oxidase, located in the hop bracts and bracteoles may have a significant role in the loss of α-acids, as it’s capable of catalysing their oxidation and can survive normal drying temperatures.8 There has also been some research indicating that hop α-acids may be indirectly oxidised or made more prone to oxidation by certain hop oil components such as myrcene and myrtenol.9 The absence of further research into these alternative degradation mechanisms is likely due to the practical inability to prevent these interactions. The hop vegetative material containing the enzyme α-acid oxidase will be present in all whole hops and pellets (reduced in T-45 pellets) while the proximity of hop oils to α-acids is unavoidable as they are both contained within the lupulin glands.

Physical factors will also influence the oxidative loss of hop resins. It’s been found that the lupulin glands, in their undamaged state provide a degree of protection to their contents from atmospheric oxygen. The lupulin gland membrane functions as a natural barrier to air, however if a large proportion of the glands are ruptured, for example during baling by the application of excessive pressure, then their protective effect will be compromised and the contents will be exposed. While higher pressure inevitably compacts the bales to a greater degree, thus limiting the rate of oxygen ingress, the overall effect is a greater rate of hop acid deterioration.10 It’s for this reasons that compressed bales are normally limited to a maximum bulk weight to preserve the intact lupulin glands and increase hop storage stability. An interesting illustration of the importance of lupulin gland integrity is that hop pellets have been found to degrade faster in air than whole hops.11 This is a direct consequence of the milling and pelletizing steps in the pellet production process, which cause damage to nearly all of the lupulin glands. The result is that pellets are particularly vulnerable to deterioration if not packaged quickly and stored in a protective environment.

Measuring changes to hop resins

Hop Storage Index (HSI)
HSI is a practical quality parameter and at present the most commonly used method for evaluating the freshness of hops or the degree to which they have degraded over time. It’s an indirect measurement of α- and β-acid decomposition and loss, that’s usually first carried out at harvest, allowing any changes to be followed thereafter. It’s a dimensionless two digit number, determined by calculating the ratio of UV absorbance at 275 nm and 325 nm of an alkaline methanol solution containing an extract of the hop being tested. The significance of the wavelengths is that absorbance at 325 nm is mainly by  α- and β-acids while at 275 nm absorbance is from unspecific decomposition products. As oxidation of the hop acids occurs, absorption at 325 nm decreases and simultaneously increases at 275 nm due to the newly formed oxidation products.

Fig. 4. Correlation between HSI and % of α- and β-acids remaining in a hop. From Peacock (1998).

HSI shows a strong correlation with the % of α- and β-acids remaining in hops (Fig. 4) and comparing the current HSI value with the value at harvest gives an indication of the % acid loss thats occurred. 12 This is a useful quality check, as it shows if proper drying, handling and storage practices have been employed. It’s also been found that a linear relationship exists between the % of α- and β-acids lost as measured by HSI and an increase in hard resin formation.13

Overall HSI is a valuable hop quality indicator but provides no information regarding the quantity or identity of oxidation products that have been formed. It’s been known for a long time that HSI and subsequent behaviour during storage is variety specific and that even when stored correctly there is usually an increase in HSI.14 It’s also been shown that the initial HSI is influenced by the hop harvest date and does not necessarily mean greater degradation has occurred.15

Lead Conductance Value (LCV)
The method for determining LCV makes use of the same principles employed to separate the hop resins. A solution of lead acetate is titrated into a hop extract / methanol solution while monitoring the conductance. The conductance value changes as an insoluble precipitate is formed with the α-acids in the extract. LCV provides a good estimate of the α-acid content of a hop and therefore it’s bittering potential. It can also be used as an indicator of how the α-acid concentration has changed with age.

High Performance Liquid Chromatography (HPLC)
HPLC can be used to measure the individual concentrations of both α- and β-acids.

Storage stability of hop resins

While the mechanisms of hop resin deterioration are poorly understood the factors affecting the rate of α- and β-acid losses in hops have been extensively investigated.

One of the more recent studies by Mikyska & Krofta found that pellets of four Czech hop varieties: Saaz, Sládek, Premiant and Agnus when stored for 12 months at 20°C in aerobic conditions, had lost between 64-88% of their original α-acid content and 51-83% of their β-acids.16 In contrast, when the same pellets were stored anaerobically at 2°C for 12 months the  α- and β-acid levels were unchanged. As the main degradation mechanism of hop resins is believed to be oxidation, it comes as no surprise that elevated temperature and exposure to atmospheric oxygen had a significant impact on their stability. The authors also observed that under anaerobic conditions, temperature had very little effect on β-acid stability whereas it did impact α-acid stability, with storage at 20°C resulting in an α-acid decline of 20–25% over 12 months. The implication being that even when hop pellets are vacuum packed, α-acid degradation can still occur if stored at around room temperature (20°C).

An investigation into the kinetics of hop resin losses by Green showed that whole hops stored at ambient temperatures in air had an initial variety dependant delay period, ranging from a few weeks to several months, during which very little loss of α- or β-acids occurred.17 After this initial lag phase it was discovered that the rate of loss of both α- and β-acids thereafter followed a first-order kinetic equation. This is interesting because the lag period implies something is preventing immediate decline. Hops have have been found to contain many potent antioxidants and it’s been suggested that their oxygen scavaging activity could go some way towards explaining this phenomenon.18

In a study of pellet hop stability by Skinner et al. it was found that Pride of Ringwood pellets showed no α-acid deterioration when stored at 5°C in either vacuum or vacuum and nitrogen atmosphere packaging for 12 months.19 At 20°C however, the α-acid loss was approximately 7% after 12 months while at 30°C it was around 40% in addition to all pellet packages displaying considerable swelling. The α-acid losses at 20°C were found to be roughly a third lower than the losses experienced with baled hops of the same variety, when stored at 22°C. The authors concluded that despite attempts to remove oxygen during pellet packing, enough precursors must have already been present to cause continued α-acid deterioration without an additional supply of air or that another degradation mechanism was involved. In addition to the loss of bittering value they recommended against storing hop pellets at ambient temperatures due to an unpleasant rancidity that developed. 

Priest et al. reported that whole Willamette hops stored between 1-4°C in air were initially stable for 11 months but that by 29 months had lost about 75% of their α- and β-acid content.20 They also showed that liquid CO2 extracts were very stable with no discernible loss of α- and β-acids, even when made using hops of varying ages. The oldest hop in the study being 17 months old when it was used to make a liquid CO2 extract. One particularly fascinating and somewhat topical finding was that when hops of varying ages were used in trial beers, the analytical bitterness or IBU was found to gradually deviate from the HPLC measured iso-α-acids. The HPLC iso-α-acids were found to be significantly less than the IBU values in beers brewer with hops of 19 and 27 months old. This is due to the less selective nature of the BU method, which also measures degradation products of the α- and β-acids. The

The main takeaways here are that storage stability can vary considerably between varieties and that the resins in both whole hops and pellets are substantially more stable when stored cold.

Products of hop acid degradation and their brewing value

We’ve seen very clearly that when hops aren’t processed or stored correctly, a sizable proportion of the α-acid content is lost. This is particularly true under aerobic conditions and at ambient temperatures (~20°C). For achieving bitterness in kettle-hopped beers the loss of α-acids in aged hops is mainly an economic issue for brewers, as a greater quantity of hops will be needed to reach the same iso-α-acid concentration. The situation becomes slightly more complicated when we begin to look at how hop resin degradation might impact the bitterness imparted from practices like dry hopping. We’re therefore interested in identifying the α- and β-acid degradation products and determining if they have any bittering value.

A variety of α- and β-acid transformation products, thought to be caused by oxidation, have been identified in hops over the years and there’s been a long history of interest in this subject with a focus on both hop and beer quality. Cook & Harris were the first to discover humulinones in 1950, believed to be products of α-acid oxidation.21 There was initially scepticism surrounding their existence among researchers with disagreement further heightened upon the detection of another group of compounds called hulupones, thought to arise from β-acid oxidation.22 Many studies have since documented compounds identified by the deliberate oxidation of individual hop acids but the real challenge has been to demonstrate that the oxidation products occur in stored hops or in beer and to isolate them. A number of recent publications have sought to re-examine the subject of α- and β-acid oxidation products using modern analytical chemistry methods like high-resolution HPLC and advanced sensory techniques. The outcome has been the confirmation that humulinones and hulupones are, as suspected, found in large amounts in degraded hops. 

Humulinones and Hulupones: Oxidised α- and β-acids

Humulinones were recently identified by Taniguchi et al. as major oxidation product of α-acids in powdered hop pellets, stored for varying lengths of time at 20°C, 40°C and 60°C while exposed to air.23 The molecular structure of humulinones (Fig. 4) is almost identical to iso-α-acids, except for an additional hydroxyl (OH) group on the 5-carbon ring. Just like the α-acids they also exist as a series of three major analogues. The hydroxyl group however makes them more polar and therefore more soluble than iso-α-acids.

Fig. 4. Oxidation of α-acids / humulones (co/n/ad) to form humulinones (co/n/ad).

In the same study, Taniguchi et al. also reported finding that hulupones were one of the main oxidation products in oxidized hops. This has since been corroborated by Dušek et al. who confirmed the presence of hulupones in Sladek hops aged for 24 and 60 months and also in the beers brewed with them.24 Interestingly they observed appreciable changes in β-acid content and composition during long term storage even though the hops were kept in anaerobic conditions in vacuum packaging. The authors didn’t however control for temperature stating that hops were “stored in conditions simulating those in unconditioned warehouses of hop merchants or breweries”, which I assume to mean a shed out the back.

Fig. 5. Oxidation of β-acids/ lupulones (co/n/ad) to form 4 hulupones (co/n/ad).

Structurally the hulupones (Fig. 5) have some similar features to their β-acid precursors, including the two characteristic 5 carbon prenyl groups. The major change from the β-acids is the transformation to a 5-carbon cyclic ring. They also exist in three main analogous forms that correspond to the original β-acid structures.

A systematic study of the bittering potential of humulinones and hulupones was conducted recently by Algazzali and Shellhammer in 2016.25 Humulinone and hulupone extracts were first prepared and dosed into unhopped lager over concentration ranges of 8 to 40 mg/L. For comparison, iso-α-acids were also dosed into the same unhopped beer with concentrations from 6 to 30 mg/L. Using a trained sensory panel they found that humulinones were on average 66% (±13%) as bitter as iso-α-acids while hulupones were found to be 84% (±10%) as bitter. They also reported that both humulinones and hulupones could be detected at the lowest concentration used in the study of 8 mg/L in unhopped beer. For reference the threshold of the more bitter iso-α-acids is around 5-6 mg/L.

Occurence of humulinones and hulupones in beer

Just to recap, up to now we’ve identified some of the major α- and β-acid transformation products in aged and oxidised hops and we know they can contribute to sensory bitterness when dosed directly into beer. What we really want to determine next is if they might have any impact when brewing.

In addition to being found in aged and improperly stored hops, research by Maye et al. showed that baled hops typically contain less than 0.3% w/w of humulinones, but their amount increased to around 0.5% w/w after the hop pelleting process and storage at 9°C.26 The same study also noted that while dry hopping with pellets, over 87% of the humulinone content dissolved in the beer within 2-3 days. They found that increasing the dry hopping dose rate (from 0-2.0 lbs/bbl) in a high-IBU beer (48 ppm of iso-α-acids, by HPLC) caused a significant drop in iso-α-acid concentration. The loss of bitterness was however, partially offset by the greater concentration of dissolved humulinones found at higher dry hopping dose rates, with humulinones contributing a noticeable bittering effect. A beer bittered entirely with humulinone extract was also compared to the same beer dosed with iso-α-acids. The bitterness profile of the humulinone beer was described as being “smoother with less clinging bitterness”. The authors also reported detecting humulinone concentrations of between 3-24 ppm (as measured using HPLC) in 29 commercial IPAs.

In a study examining beer bitterness and the effect of hop bitter acids, Oladokun et al. reported finding humulinone concentrations of ≤ 3 mg/L in 34 commercial lagers from around the world.27 This is in contrast to a more recent study in which Hahn et al. found detectible levels of humulinones (≥1 mg/L) in 117 out of 121 unique commercial beers.28 The beers profiled included a mixture of hoppy ales and lagers with the average humulinone concentration reported to be 17 mg/L. On the other hand, when testing for the presence of lupulones and their oxidation products such as hulupones, the authors found they were either not detectible or present at levels so low they couldn’t be quantified.

On this evidence it would seem that humulinones may have a more important bittering role than hulupones but research in this area is far from conclusive. What still isn’t clear is if a greater quantity of these potential bittering substances are imparted to beers, either through conventional kettle-hopping or dry hopping, when aged or oxidised hops are used. I’ve also touched upon just a fraction of the α- and β-acid degradation products that have been identified and barely mentioned the hard resins, all of which may be revealed to have more importance in the future. It’s at this point that the trail goes cold and we’ll have to wait a bit longer for science to give use more answers I’m afraid.

References

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