Carlos Silva Ferreira’s research while affiliated with Université Catholique de Louvain - UCLouvain and other places

Dry hopping imparts distinct aromas but also a series of non-volatile compounds suspected of causing flavor and physical instability during beer storage. In this work, color, chill haze, total polyphenols, total flavanoids, and flavan-3-ol monomers (catechin and epicatechin) and oligomers (procyanidin dimers and trimers) were monitored in five commercial pale-colored Belgian dry-hopped beers over 24 months of storage at 20 °C in the dark. Fresh dry-hopped beers contained unusually high levels of flavan-3-ol monomers (up to 6.6 mg/L) and oligomers (up to 14.1 and 10.2 mg/L dimers and trimers, respectively). The increase in color intensity during storage (up to 6.4°EBC) correlated with fresh beer monomer levels, while the oligomer content correlated with chill haze formation (up to 25.7°EBC). The evolution of these two physical attributes also correlated with the level of total polyphenols in the fresh beers. In a pilot-scale production, kettle hopping was shown to impart either monomers (early) or oligomers (late), while dry hopping promoted efficient extraction of both monomers and dimers (extraction yields of 62 and 74%, respectively). Dry hopping thus plays an important role in color and chill haze increase.

The aim of the present work was to compare levels of short chain fatty acids, esters, terpenoids and polyfunctional thiols in (mostly bottle-refermented) commercial Belgian dry-hopped beers before and after 2 years of storage at 20 °C (the usual best-before date in Belgium). Among the hop-derived volatiles, the terpenoids linalool and geraniol, the polyfunctional thiols 3SHol, 3SHA and 3S4MPol, and the esters ethyl isobutyrate, ethyl isovalerate and ethyl heptanoate (up to 499, 53, 0.2, 2, 3, 84, 63, and 19 µg/L, respectively) were found above their sensory thresholds in most fresh dry-hopped beers. The fermentation-derived esters reached concentrations similar to those previously reported for non-dry-hopped beers, with ethyl hexanoate and isoamyl acetate (up to 0.4 and 3.9 mg/L, respectively) often above their sensory thresholds. Except ethyl isovalerate (more than 85% still present), most hop odorants and fermentation esters showed degradation over the 2-year storage period: only 45%–70% of linalool, geraniol, and ethyl hexanoate and even less than 40% for polyfunctional thiols, ethyl isobutyrate, and ethyl heptanoate initial concentrations were detected after storage. How the dry-hopping process affects this degradation was further investigated in model media. Fermentation esters proved to be more strongly impacted in dry-hopped than in non-dry-hopped beers because of hop esterase activity. In addition to being aware of the need to avoid hop esterases, craft brewers are here advised to use bottle refermentation for its ability to regenerate some flavors and consume packaged oxygen. No deleterious effect of yeast, such as short chain fatty acid excretion, was evidenced.

The evolution of isohumulones, main bitter compound of beers, through storage and its bitter impact has been studied for many years. With the uprising of highly hopped and, especially, dry-hopped beers during the last decade, other compounds such as humulones, cis-humulinones and hulupones have been shown to participate in both the sensory and measured bitterness of these beers. However, the fate of these compounds through aging is still unclear. The present work evaluated, by means of spectrophotometric and RP-HPLC-UV analyses, the evolution of bitterness units (BU), cis- and trans-isohumulones, humulones, cis-humulinones, hulupones, and tetrahydro-isohumulones in 21 Belgian dry-hopped beers after two years of storage. Besides the expected degradation of trans-isohumulones, it was observed an average decrease of 91% of humulones and 73% of cis-humulinones. Together, these results indicate a decrease of 18 – 43% of the calculated bitterness intensity of dry-hopped Belgian beers. The losses of cis-humulinones in dry-hopped beers bring seven new oxidation compounds, some of them very similar to those issued from trans-isohumulones. The bitterness impact of these compounds is yet to be determined.

The aromatic complexity of craft beers, together with some particular practices (use of small vessels, dry hopping, etc.), can cause more oxidation associated with pre-maturated colloidal instability, Madeira off-flavors, bitterness decrease, and aroma loss. As bottle refermentation is widely used in Belgian craft beers, the aim of the present work is to assess how this practice might impact their flavor. In fresh beers, key flavors were evidenced by four complementary techniques: short-chain fatty acids determination, esters analysis, XAD-2 extract olfactometry, and overall sensory analysis. In almost all of the fresh beers, isovaleric acid was the sole fatty acid found above its sensory threshold. Selected samples were further analyzed through natural aging at 20 °C. The presence of yeast in the bottle minimized the trans-2-nonenal released from Schiff bases and proved less deleterious than suggested by previous studies with regard to fatty acid release and ester decrease through aging. Furthermore, according to the yeast species selected, some interesting terpenols and phenols were produced from glucosides during storage.

Although long renowned worldwide for its unique dry-hopped (DH) Trappist beer, Belgium did not develop this process for other brands until the last decade. Twenty-one commercial Belgian DH beers were investigated and compared with a few other typical Belgian beers whose production involves either late hopping or aged hop addition (Gueuze). Bitterness was determined by spectrophotometric measurements (isooctane extraction) and by reversed phase high performance liquid chromatographic with UV detector (RP-HPLC-UV) (simultaneous quantitation of humulones, cis-/trans-isohumulones, reduced isohumulones, humulinones, and hulupones). In dry-hopped Belgian beers, humulinones (found at concentrations up to 13.3 mg/L) were estimated to be responsible for up to 28% of their bitterness. As humulinones revealed to be gradually lost through boiling (22%), clarification (5%), and fermentation (14%), non-dry-hopped (NDH) beers often displayed levels below 1.7 mg/L. Even in Gueuze beers for which old, humulinone-containing hops are used, no humulinone was found. Contrary to humulones, which were detected up to 7.2 mg/L in DH beers, hulupones were found at less than 3 mg/L in all Belgian beer styles. Humulinones were not produced in the boiling wort from humulones (in contrast to hulupones, readily synthesized from lupulones) but were significantly solubilized from hop thanks to their hydrophilicity. Yet, while the co-form accounted for about 50% of the humulones, the n-form prevailed for humulinones. Some humulinone degradation products were evidenced by RP-HPLC-MS/MS, and as suggested by their retention time (RT), should be more polar than their precursors. Bottle refermentation emerged as an additional critical step of humulinone loss, explaining the low levels found even in some strongly DH beers.

The dual-purpose hop varieties Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace were recently shown to contain unusually high amounts of some discriminating terpenoids, polyfunctional thiols, and precursors of the latter (cysteine and glutathione adducts). The present work aimed to investigate the terpenol glucoside fraction in hops and its potential contribution to beer after a dry hopping process. Terpenols were quantified by stir-bar sorptive extraction GC-MS in five pilot monovarietal dry-hopped beers. In all of them, linalool and geraniol were found above their sensory thresholds (72-178 and 7-57 μg/L, respectively, for a threshold of 8 μg/L for linalool and 4 μg/L for geraniol). β-Citronellol also exceeded its threshold when the Amarillo, Citra, or Sorachi Ace cultivars were used. The hop glucoside potential was analyzed by GC-MS after enzymatic degradation. A relative hydrolysis efficiency factor was applied to our data to take into account that the commercial P-glucosidase releases octan-1-ol, used here as an internal standard, 2.8 times more efficiently than geraniol. β-Glucosidase treatment caused the release of linalool, α-terpineol, β-citronellol, and geraniol from all five dual-purpose cultivars, but in much lower amounts than the corresponding free terpenols (0.6-28.6 mg/kg of aglycons versus 7.8-109.2 mg/kg of free forms). Further quantitative analyses focusing on more traditional aromatic and bitter hops are now needed to compare their glucoside fractions with those here investigated.