Kappler Et Al-2010-Journal of the Institute of Brewing

332 JOURNAL OF THE INSTITUTE OF BREWING

Degradation of Iso-α-Acids During Wort Boiling

S. Kappler1,*, M. Krahl1,2, C. Geissinger1, T. Becker1 and M. Krottenthaler1

ABSTRACT

J. Inst. Brew. 116(4), 332–338, 2010

A detailed study on the degradation of iso-α-acids was con-ducted. Because of the complexity of the wort matrix and inter-fering interactions during real wort boiling, the investigation of degradation kinetics was performed in an aqueous solution. Deg-radation was investigated as a function of time (0–90 min), tem-perature (80–110°C), pH value (4–7), original gravity (10–18°P) and ion content of the water (0–500 ppm Ca2+ and Mg2+). After 90 min of boiling, over 20% of the dosed iso-α-acids could no longer be detected. A strong dependence of degradation could be shown due to high temperature, low pH, high original gravity and a high Mg2+ content. The cis:trans ratio and co-iso-α-acid content did not change significantly. Losses of isohumulones could be lowered by reducing the temperature and original grav-ity, as well as by heightening the pH value. High amounts of Ca2+ and Mg2+ salts also led to an increase in degradation prod-ucts. Solutions to decrease degradation and thereby possible improvements in sensory bitter quality are discussed.

Key words: Hops, iso-alpha acids, wort boiling, utilisation rate.

INTRODUCTION Hops, used in brewing, are the dried cones of the fe-

male plant Humulus lupulus sp. Other than in some spe-cialized beers, only unfertilized cones are used. The beginning of the use of hops in brewing is believed to date back to the 11th or 12th century. It probably took place in different regions of Belgium and Germany. The growing popularity of hops is believed to be due to the bacterio-static properties of the α- and iso-α-acids16,26. Thus beers brewed with hops were more stable against infections and negative health effects occurred less often when hops were used. The lower amount of infected beers was surely one of the reasons why hops were the only allowed spicy adjunct in the brewing process4. The most popular decla-ration regarding brewing is the Bavarian Purity Law, en-forced in 1516, which defined hops as the only spicy ad-junct allowed in beer. Nowadays, in over 99% of all brews, hops are the only spice used43.

Hop bitter acids are the main bitter compounds in beer. They contribute to more than 85% of the overall bitter-ness35. Humulones, also called α-acids, which are present in hops undergo an isomerisation reaction to isohumu-lones, also known as iso-α-acids, after thermal treatment2. At least five homologues of humulones exist and these only differ in one side chain at the C2-position51.

The percentage of the three main homologues, co-, n- and ad-humulone, is around 90%. During the isomerisa-tion reaction, two stereoisomers, cis- and trans-isohumu-lone are formed from each of the homologues50 and these differ in their properties. It is known that cis-isohumulone is more stable against ageing, but has weaker foam stabi-lising properties20. Previously published work shows that a mixture of cis- and trans-isohumulones is more bitter in beer than trans-isohumulones alone1. It is also known that pure cis-isohumulones are more bitter than trans-isohu-mulones21. Just recently, purified cis-isohumulones were isolated for the first time28. Losses during the fermentation and storage of beer were shown to be higher for trans-isohumulones than for cis-isohumulones8, although there is little difference in polarity between the cis- and trans-isohumulones20. Jaskula et al.25 found that iso-α acid for-mation followed first-order kinetics and Arrhenius behav-iour. Differences in activation energy for the formation of trans- and cis-isomers were measured. The activation en-ergy for the formation of trans-iso-α acids was approxi-mately 9 kJmol–1 lower. Usually the yield of iso-α acids derived from the dosed hops is not more than around 30%38. This low yield is affected by numerous factors. One of the main reasons is the poor solubility of the uni-somerised humulones, as determined by Kolbach29 and Spetsig49. Solubility of α-acids can be improved by using an increased pH value, which may result in an accelerated isomerisation reaction, although the isomerisation reac-tion itself is not affected by changes in pH33. Additionally, boiling time, boiling temperature, original gravity, type of hop product and hop variety used are all factors that influ-ence the yield of iso-α acids13,33–34,41. Prolonged boiling time, a higher temperature and a higher concentration of α-acids in the dosed product have all shown a positive effect11,17,18. Higher extract levels and higher concentra-tions of α-acids in wort have resulted in lower yields34. Another factor is the content of divalent cations and their salts in the reaction medium. Some of these, for example calcium and magnesium salts, were found to promote the isomerisation of α-acids into iso-α acids in higher amounts31. The use of pre-isomerised hop products such as isomerised kettle extract or iso-pellets is a possible approach to achieve higher yields in the cooled wort. However, losses during fermentation and filtration are reasons for a poor recovery rate of isohumulones in the finished beers19,32,36. These losses are due to adhesion onto

1 Technische Universität München, Lehrstuhl für Brau- und Getränketechnologie, Weihenstephaner Steig 20, 85354 Freising-Weihenstephan, Germany.

2 MEG, Langendorfer Straße 23, 06667 Weißenfels, Germany. * Corresponding author. E-mail: [email protected] Parts of this paper were presented at the 2nd International Sympo-sium for Young Scientists and Technologists in Malting, Brewingand Distilling, May 19–21, 2010 in Freising – Weihenstephan, Ger-many.

Publication no. G-2010-1206-117 © 2010 The Institute of Brewing & Distilling

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yeast cells and filter aids. Carbon dioxide as the primary yeast metabolite, in addition to ethanol, removes hop derived compounds from the fermenting medium32. A further approach to reduce losses is the use of downstream products. Besides the traditionally used iso-extract, which is added just before filtration, the use of reduced isohu-mulones such as rho-, tetrahydro- or hexahydro-isohumu-lones can reduce the risk of production of a skunky light-struck flavour in bottled beer and also improve foam stability27.

Some knowledge exists about the degradation kinetics of humulones during hop storage7,42. Many substances are formed from α-acids by various chemical reactions51. These substances are known to partly survive the brewing process or are converted during wort boiling to com-pounds which impart a bitter flavour to beer44. Several papers have been published regarding the factors that in-fluence the stability of α-acids during storage and the in-fluence of the degradation products on the brewing quality of the hops7,9,10,40,54. The “Hop Storage Index” (HSI) is used as an indicator of hop freshness42,52. It has been reported that a small amount of degradation products can improve the quality of the bitterness in beer15. In larger amounts, possibly resulting from the alkaline isomerisa-tion of α-acids, the sensory impression can be negatively influenced due to the formation of humulinic acid14. Recent research has also shown that there are some degra-dation reactions which are specific for trans-isohumu-lones22,23. These reactions form substances with a linger-ing and a harsh bitter taste33.

The role of iso-co-humulones has been controversial in the past. Schönberger47 summarised as follows: By in-creasing pH value, more of the isohumulones are dissoci-ated. This can lead to an unpleasant and harsh bitterness in beer6,37. Rigby45, who compared beers with different amounts of isohumulones, stated that iso-co-humulones create a harsher and more intense bitterness. Newer re-search has contradicted this finding and could not find a negative effect of iso-co-humulone to bitter qual-ity30,39,48,53. Furthermore, there was a higher yield due to the higher polarity and thus better solubility47. Neverthe-less, many brewers still favour a low iso-co-humulone content in the finished beer.

Recent research has shown that nearly 25% of the dosed α-acids cannot be found in the trub or hot wort, either as α-acids or as iso-α acids12. Little knowledge ex-ists about their fate. The aim of this study was to gain an insight into the degradation process of the isohumulones during thermal treatment. Changes in iso-α-acids were monitored in a model solution during a simulated wort boiling process.

MATERIALS AND METHODS Samples and experimental setup

Pre-isomerised pure iso-alpha acids (Barth-Haas Group, Nürnberg, Germany) were diluted in distilled wa-ter, lauter wort or diluted unhopped malt extract (Weyer-mann, Bamberg, Germany) respectively. The concentra-tion of the isohumulones was 100 ppm in all trials. In order to simulate wort boiling, the samples were heated in an oil bath (Memmert, Schwabach, Germany) filled with

polyethylene glycol (BASF, Ludwigshafen, Germany) using an inert reflux condenser. The parameters of time, original gravity, pH, and temperature were varied. Boiling time was set between 15 and 90 min with intervals of 15 min. Original gravity was between 10 and 18°P with steps of 1°P. Temperature was set to 80°C, 90°C, 100°C and 110°C respectively. The pH value was adjusted before boiling to 4.0, 5.0, 6.0 and 8.0 using phosphoric acid (Baker, Deventer, The Netherlands) or sodium hydroxide (Baker, Deventer, The Netherlands). Unless indicated oth-erwise, boiling time was set to 60 min. The effect of water hardness was investigated using calcium sulphate (Sigma-Aldrich, Schnelldorf, Germany), magnesium sulphate (Sigma-Aldrich, Schnelldorf, Germany) and calcium chlo-ride (Sigma-Aldrich, Schnelldorf, Germany) in concentra-tions of 200 and 500 ppm and referred to Ca2+ and Mg2+. The influence of different lauter fractions was determined in separate boiling trials according to Yamashita et al.55 Iso-α-acids levels were measured before and after the boiling process. All trials were carried out in duplicate. All measurements were checked for suitability with the coefficient of variation.

Determination of iso-α-acids

Iso-α-acids were measured after extraction with iso-oc-tane (Baker, Deventer, The Netherlands) according to the method presented by Jaskula et al.24 with some minor modifications. Separation of the homologues and stereoi-somers was performed using an Alltech Alltima C18, 5 µm, 150 × 4.6 mm column (Grace, Columbia, MD, USA). The liquid chromatographic system consisted of an Agilent 1100 series system with autosampler, quaternary pump, thermostat and an Agilent 1200 series diode array detector (DAD). Isocratic elution was conducted with 52% acetonitrile, HPLC gradient-grade (Baker, Deventer, The Netherlands) and 48% of bi-distilled water (Milli-pore, Billerica, MA, USA) adjusted to a pH of 2.8, with phosphoric acid (Baker, Deventer, The Netherlands). In-jection volume was 25 µL and time of analyses was set to 70 min. The thermostat was set to 30°C. Because brown vials had shown lower retrieval rates in previous trials due to iron traces in the glass5, clear vials were used. Detec-tion wavelength was set to 270 nm. Prior to analysis, the samples were filtered through a 0.45 µm membrane filter.

Calibration was conducted using ICS-I2 (Labor Veri-tas, Zürich, Switzerland) as the external standard. The coefficient of variation for this analysis was below 2% (data not shown).

RESULTS AND DISCUSSION During wort boiling at atmospheric conditions, a con-

stant decrease of isohumulones was observed (Fig. 1). After 30 min of boiling, the recovery of isohumulones was about 90%. Subsequently a linear drop to a recovery rate of 77% after 90 min was measured. The cis-trans ratio showed a slight increase from 4.5 to 4.7 in this trial (data not shown). Regarding the amount of co-isohumu-lones, no significant effect of boiling time was seen.

Figure 2 shows the effect of different temperature treat-ments over 60 min on the recovery rate of the isohumu-lones. At 80 and 90°C, isohumulone losses of 5 and 9%


were measured. During atmospheric boiling, losses were more pronounced and only 84% of the dosed isohumu-lones were recovered. A further increase in temperature led to a recovery rate of only 81%. The cis-trans ratio changed from 4.5 to 4.8 (data not shown). No changes were seen regarding the iso-co-humulone fraction.

Figure 3 shows the effect of pH changes on the recov-ery rate of isohumulones after boiling. At a pH of 4.0, only 58% of the initially dosed isohumulones were found.

Detection rate was 80% at a pH of 5.0, 86% at a pH of 6.0 and 95% at a pH of 8.0. No changes were seen in the cis-trans ratio, while the co-isohumulone fraction decreased slightly from 48% at a pH of 4.0, to 45% at a pH of 5.0, to 44% at a pH of 6.0 and to 41% at a pH of 8.0.

Figure 4 shows the effect of varying the original grav-ity on the recovery rate of isohumulones after wort boil-ing. The recovery rate dropped linearly from 90% at 10°P to 52% at 18°P. No changes were observed in the cis-trans

Fig. 1. Recovery rate of dosed isohumulones during 90 min of atmospheric boiling.

Fig. 2. Recovery rate of dosed isohumulones after treatment with different temperatures.

Fig. 3. Recovery rate of dosed isohumulones after atmospheric boiling with different pH values.

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ratio. The relative amount of iso-co-humulones increased with increasing original gravity from 38% at 10°P to 45% at 18°P (Fig. 5).

These results were confirmed by using different lauter fractions during the boiling trial. In the first wort, only 48% of the isohumulones initially present were detected. Boiling of the isohumulones with the last running resulted in an 83% recovery. Iso-co-humulone levels were 45% in

the first wort, and decreased to 38% in the last runnings. Addition of mineral salts resulted in lower recovery

rates. Compared to a recovery of 83% after boiling with distilled water, values were 38% lower after the addition of 200 ppm CaCl2 and 48% lower after the addition of 500 ppm CaCl2. Addition of 200 ppm MgCl2 resulted in a decrease of 52%. Boiling with 500 ppm MgCl2, resulted in a 54% lower recovery rate. Boiling with CaSO4 re-

Fig. 4. Recovery rate of dosed isohumulones after atmospheric boiling with varying original gravity.

Fig. 5. Relative co-isohumulone content after atmospheric boiling with varying original gravity.

Fig. 6. Recovery rate of dosed isohumulones after atmospheric boiling with varying ion content.


sulted in 10% lower isohumulone levels at 200 ppm and 25% lower levels at 500 ppm (Fig. 6). The cis-trans ratio decreased in all trials (Fig. 7).

However, this decrease was only significant after the addition of MgCl2. In this particular case, the ratio de-creased from 4.7 to 3.8 after the addition of 200 ppm and to 3.6 after the addition of 500 ppm. Addition of chloride containing salts did not significantly change the percent-age of the iso-co-humulones. Levels increased from 38% to 39% with a dosage of 200 ppm, and to 41% with 500 ppm CaCl2. In the boiling trial with MgCl2, 41% of the iso-co-humulones could be measured after an addition of 200 ppm and 43% after an addition of 500 ppm. No effect was seen after dosage with CaSO4.

The results obtained in these trials demonstrated that prolonged wort boiling, as well as higher original gravity, led to lower final concentrations of isohumulones. The higher temperatures and lower pH values, as well as the dosed mineral salts, also lowered the recovery rate. In the practical brewing process, lower temperatures during wort boiling and treatment (e.g. temperatures in the heat ex-changer) will reduce the losses of the isohumulones and lead to a lower amount of undesired degradation products in the cooled wort. Regarding the degradation of isohu-mulones, whirlpool temperature should not exceed 90°C. Simultaneously, lower temperatures offer further advan-tages such as lower total DMS levels, as DMS-P cleavage is reduced3. If iso-kettle-extract is being used during wort boiling, a short boiling time after dosage should be chosen to guarantee an optimum isohumulone yield. Late addi-tion of iso kettle extract, is not a problem regarding the yield of isohumulones, as no further isomerisation reac-tion is needed. However, although isomerisation is already completed, the evaporation of undesired aroma compo-nents is needed. Consequently, at least a short boil is rec-ommended. Additional wort acidification should be per-formed just before the end of the wort boil to reduce degradation of the isohumulones. This beneficial effect of late acidification is independent of the hop product used. Regarding water treatment, the usage of calcium sulphate is beneficial towards a higher yield of isohumulones, in comparison to calcium and magnesium chloride. In the brewing process, calcium sulphate and calcium chloride, as well as magnesium chloride lead to a lower pH during

mashing and are thus beneficial for the desired enzymatic activities and can reduce undesired enzymatic activities46. Consequently, brewers need to compromise between beneficial effects during mashing, and the degradation of isohumulones during the wort boil, if chloride containing salts are used. Regarding high-gravity brewing, the results indicated that higher losses are to be expected during the wort boil. A possible approach to reduce such losses, es-pecially if iso kettle products are being used, might be a separate boiling of the hops with the last runnings. This would limit losses and optimize recovery rates. However if traditional hop products are used another problem has to be taken into account. On the one hand, the unisomer-ised α-acids from hops, pellets or hop extract have to be dissolved and subsequently isomerised. On the other hand, degradation of the isohumulones is not desirable and should be limited to a minimum. Equilibrium be-tween isomerisation and degradation must be found to guarantee optimum yields. If hops are added at the begin-ning of wort boiling, the degradation reaction could lead to an increase in less favourable flavour impressions un-der unfavourable conditions.

CONCLUSIONS In this work, boiling trials were performed to evaluate

the degradation kinetics of isohumulones, the main bitter-ing substance in beer. Isohumulones, which are formed from hop-derived humulones, are not stable under thermal treatment. Various degradation products form and these can create a harsh bitterness and lingering aftertaste. To-day many of these substances have been identified, but the kinetics of their formation is still unknown. The aim of this study was to evaluate the factors affecting the degra-dation of the isohumulones. Boiling trials on a lab-scale with purified iso-α-acids, at a concentration of 100 ppm, were conducted. Temperature, pH value, original gravity, lauter fraction and ion content were varied. Before and after the thermal treatment, the content of the isohumu-lones was measured. The recovery rates of the isohumu-lones, as well as the cis:trans ratio and the relative amount of iso-co-humulone were calculated.

By varying the wort composition and the boiling pa-rameters, losses of isohumulones during wort boiling and

Fig. 7. The cis:trans ratio after atmospheric boiling with varying ion content.

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wort treatment could be reduced. Boiling at atmospheric pressure for 90 min, comparable to a standard boil and hot wort treatment, led to losses of nearly 25% within 90 min. These losses could be minimized by lowering the tem-perature. High losses were also found when isohumulones were boiled at a low pH value, high original gravity and high water hardness. By optimizing these parameters, losses of isohumulones and thereby a decrease in less favourable bitter impressions can be attained.

ACKNOWLEDGEMENTS

This work was supported by the Barth-Haas Group.

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55. Yamashita, H., Kühbeck, F., Hohrein, A., Herrmann, M., Back, W. and Krottenthaler, M., Fractionated boiling technology: wort boiling of different lauter fractions. Monatsschr. Brauwiss., 2006, 64, 130-147.

(Manuscript accepted for publication August 2010)

Documents

Kappler Et Al-2010-Journal of the Institute of Brewing