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Why Catechin and Epicatechin from Early Hopping Impact the Color of Aged Dry-Hopped Beers while Flavan-3-ol Oligomers from Late and Dry Hopping Increase Colloidal Instability
Abstract
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 Journal of the American Society of Brewing Chemists occupied the second position, with 22 published documents and 295 citations (H-Index 45 and impact factor of 2.88 for the period 2022-2023). This position was achieved due to the contribution of several researchers, highlighting the work by Silva Ferreira et al. [36], who stated that the amber color of pale-colored Belgian beers is influenced by the polyphenol content (epicatechin and catechin) during storage, as well as by the dry hopping process. [26] and Coghe et al. [27], which are among the ten most cited (Table 1), and Dugulin et al. [35]. ...
... The Journal of the American Society of Brewing Chemists occupied the second position, with 22 published documents and 295 citations (H-Index 45 and impact factor of 2.88 for the period 2022-2023). This position was achieved due to the contribution of several researchers, highlighting the work by Silva Ferreira et al. [36], who stated that the amber color of pale-colored Belgian beers is influenced by the polyphenol content (epicatechin and catechin) during storage, as well as by the dry hopping process. ...
... In conventional beers, color is derived, in large part, from malted grain [38]. Phenolic compounds (polyphenols) derived from barley husk or hops are secondary metabolites of plants and may also contribute to beer color [21,36,52,53]. Besides malt and polyphenols, some additives, such as caramel colorants added to the final formulation and syrups, also contribute to the color of beer [7,21]. ...
Beer is a popular beverage consumed globally, and studies have emphasized the benefits of moderate consumption as well as its sensory effects on consumers. Color is a crucial sensory attribute, being the first aspect a consumer notices when assessing a beer’s quality. This review seeks to offer detailed insights into how brewing methods, raw materials, and the chemical diversity of beer influence the production of beer color. The chemical mechanisms responsible for color development and how consumers and color systems perceive the color of beer were assessed. A systematic review following the PRISMA methodology, coupled with a bibliometric analysis, was performed using (Rayyan 2022) and (VOSviewer 1.6.20) software to assess and evaluate the scientific research retrieved from the Web of Science Core Collection. The findings highlight the significant roles of malt types, heat brewing processes, control of chemical parameters, and innovative brewing techniques in conventional beer color production. Novel chromophores like perlolyrine, pyrrolothiazolate, and furpenthiazinate are thought to affect Pilsen-style beers, along with melanoidins, Strecker aldehydes, and 5-hydroxymethylfurfural (HMF) in conventional beers. In craft beers, such as fruit- or herb-based beers, flavonoids like anthocyanins, along with other natural pigments and synthetic colorants, are identified as the primary sources of color. However, studies related to the influence of chromophores like perlolyrine, pyrrolothiazolate, and furpenthiazinate on beer color are scarce, and emerging additives, such as pigments from microorganisms, spices, exotic herbs, and leaves of plants, on craft beer offer insights for future research.
... Therefore, in most cases, the total polyphenol content of a beer correlates directly (R 2 = 0.8) with its antioxidant activity (contribution of 55-88%) [1,8,31,36]. Unfortunately, oxidation products derived from polyphenols can also negatively affect color and colloidal stability [8,37,38]. During fermentation, yeast also produces antioxidants, mainly sulfites (through the conversion of sulfates, methionine, or cysteine) and glutathione [3,8,26,39]. ...
... Other antioxidants present in beer at very low levels include carotenoids and tocopherols [31,32,40], saponarin, and hordatines A-C [8,41]. Some additional antioxidants may come from dry hopping [37], spices/herbs (e.g., hibiscus, juniper, lemon balm, etc. [42,43]), fruits (e.g., cherry juice and goji berry [44,45]), flavorings and colorings [7], or alternative raw materials (e.g., sorghum and buckwheat [46,47]). ...
... Trolox/L) because of its dry hopping process. Hop is known to show a 30 times greater intrinsic antioxidant capacity than pale malt [25,55], thanks to its very high level of polyphenols [37,55,56]. The total polyphenol content, as already shown by other studies [8,41,57], appeared to contribute most to the antioxidant power of each beer (42-100%; Table 1), with a major proportion attributed to flavan-3-ols (catechin ORAC value = 11.2 µmol eq. ...
Even when fresh, non-alcoholic, and low-alcoholic beers (NABLABs) exhibit significant staling defects due to premature oxidation. In this study, the antioxidant power of eleven fresh commercial NABLABs was assessed by means of three different assays: the oxygen radical absorbance capacity (ORAC), the linoleic acid-induced oxidation (TINH), and the indicator time test (ITT). Only the first two assays, both involving radicalar degradations initiated by AAPH, were found to correlate with each other. NABLABs displayed lower ORAC values than conventional beers (on average, 6127 μmol eq. Trolox/L), except for three samples made with special-colored malts or dry-hopped. Dealcoholization was the step with the greatest impact on the ORAC value (up to a 95% loss) and on flavan-3-ols, sotolon, and polyfunctional thiols, while pasteurization strongly affected color, TBA, and Strecker aldehydes. ORAC assays applied to hop, alternative cereals, and various botanical ingredients indicated that mashing with red sorghum, dry hopping/spicing, and wood maturation could bring the antioxidant power of a NABLAB close to those of conventional beers. With an ORAC value not reached by any other tested botanical ingredient (5234 µmol eq. Trolox/g), African Vernonia amygdalina leaves (traditionally used for Rwandan Ikigage beers) emerged here as the best candidate.
... The polyphenols involved in beer haze and color are mainly oxidation products of mono-, di-and trimers of flavan-3-ols (various combinations of catechins and epicatechin, as illustrated in Figure 1), a subgroup of flavanoids (i.e., flavonoids with a flavan-heterocycle) with an OH in position 3 [8][9][10][11]. Recent studies have shown that dehydrodi(epi)catechin A (from (epi)catechin monomer oxidation) can modify beer color [12]. On the other hand, complexes resulting from interactions between A-type dimers (from procyanidin oxidation, requiring epicatechin as the first subunit) or oligomers and malt hordein are responsible for colloidal haze [13][14][15][16]. ...
... On the other hand, complexes resulting from interactions between A-type dimers (from procyanidin oxidation, requiring epicatechin as the first subunit) or oligomers and malt hordein are responsible for colloidal haze [13][14][15][16]. While early hopping will enhance depolymerization and thus increase monomer concentrations, causing a color intensity increase during storage, late and dry hopping, through simple solubilization, will rather increase dimer and longer oligomer levels, creating colloidal instability in aged beers [12,17]. In non-alcoholic and low-alcoholic beers (NABLABs), among which hop-forward products have become famous thanks to the masking of the worty off-flavor, the lack of ethanol leads to premature oxidation. ...
... Based on already published beer polyphenol analyses [12], flavan-3-ol extracts were purified on Sephadex LH-20 resin (a well-known resin used for high polyphenol recovery [38]), yet after hop delipidation and solid-phase extraction steps (adapted from Jerkovic et al. and Li et al. [33,39]). The purified extracts were analyzed by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) with ESI(-)-MS/MS detection [12]. ...
While the Saaz aromatic variety remains classified as a total-flavanoid-rich cultivar, no inverse correlation was found between total flavanoids and the α-acid content when the dual-purpose varieties Citra, CTZ, Amarillo, Eureka!, Mandarina Bavaria, Mosaic, Polaris, and Sabro were considered. The levels of hop flavan-3-ol monomers, dimers, and trimers (quantitated by HPLC-MS/MS) appeared strongly influenced by variety and harvest year. On the other hand, the catechin/epicatechin ratio (and B3/B2 ratio) proved stable within the same variety through two successive harvest years. Among the nine herein-investigated varieties, Citra and Saaz displayed notable catechin/epicatechin ratios (>3.7 compared to <1.6 for the others), whereas Polaris exhibited the lowest monomer content (less than 800 mg/kg). These distinctive profiles could impact the colloidal and color stability of hop-forward beers.
... As a good radical scavenger of hydroxyl radicals (HO°) and other reactive oxygen species ROS (e.g., O 2°− , HOO° issued from metal-induced Fenton and Haber-Weiss reactions [12][13][14][15][16]), it can prevent volatile and non-volatile compounds to be oxidized during storage in regular beer. Polyphenols and bitter compounds are recognized to be particularly sensitive to degradation during beer storage, especially in dry hopped beers [17][18][19][20][21]. In NABLABs, we can suspect that they will be still less protected in the absence of radical scavengers. ...
... Then, flavan-3-ols and bitter compounds levels were determined by Reverse-Phase High-Performance Liquid Chromatography (HPLC) with ESI(-)-MS/MS and UV detections. The fate of these attributes was further briefly compared to similar data recently published for the top dry hopped fermentation beers [18,21]. ...
... Chill haze and color are known to be oxidation markers of a beer. As recently shown for dry hopped beers [21], catechin and epicatechin mainly impact color (synthesis of dehydrodi(epi)catechins A by oxidation), while flavan-3-ols oligomers (dimers and trimers, especially those including an epicatechin fragment) increase colloidal instability (synthesis of A-type oligomers by oxidation). Spectrophotometric global methods were first applied to assess the fate of polyphenols. ...
Nowadays, non-alcoholic (NAB) and low-alcoholic beers (LAB) still significantly suffer from staling defects when fresh, partially due to absence of ethanol as antioxidant. In the current work, the fate of flavan-3-ols (monomers, dimers, and trimers) and bitter compounds (isohumulones, humulinones, etc.) of 11 commercial NABLABs available on the Belgian market was monitored through one year of aging at 20 °C in the dark. Fresh NABLABs contained variable flavan-3-ols and bitter compounds levels (between 3.0–10.0 mg/L and 8.0–39.0 mg/L, respectively), depending on different technological processes used. Chill haze and color were also investigated as potential oxidation markers of fresh and aged beers. Surprisingly, contrary to conventional beers, the oligomers’ concentration (dimer and trimer procyanidins) exhibited a strong correlation (R2 = 0.95) with chill haze before aging, suggesting prematured oxidation of the samples. After a year of storage, significant degradation occurred as for regular dry hopped beers (process very sensitive to oxidation), only 27% remaining for flavan-3-ol dimers and an average 16% for trans-isohumulones. Oxidation risk appears here as the main weakness of NABLABs, which could be probably improved by spiking very efficient antioxidants.
... Future studies should as well investigate residual trace element and polyphenol composition in spent hops, as reported concentrations in hops and transfer rates during dry hopping vary. [32,37,39,[59][60][61][62][63] This may influence the quality of spent hops with regard to oxidative and haze stability or donor for auxiliary bitter compounds when re-used. [59][60][61]63] In the current study, HPLC analysis was available to quantify humulone in spent hops and therefore allowed to calculate spent hop additions precisely. ...
... [32,37,39,[59][60][61][62][63] This may influence the quality of spent hops with regard to oxidative and haze stability or donor for auxiliary bitter compounds when re-used. [59][60][61]63] In the current study, HPLC analysis was available to quantify humulone in spent hops and therefore allowed to calculate spent hop additions precisely. HPLC analysis is not widely used in brewing quality control and methods allowing a simple estimate of the humulone content in spent hops would be favorable. ...
... The differences in color change in beers dry hopped with different hops varieties may be due to chemical composition of the hops (for example, polyphenols that pass into beer during dry hopping, and determines the course of beer oxidation, which may be manifested by its darkening). [28] The higher increase of beer color in pasteurized beers may be explained by the absence of living yeast cells that if present, would rapidly use up any oxygen picked up due to dry hopping. [11] Consequently, the pasteurized beers after dry hopping can get oxidized and thus get darker. ...
Abstract: Dry-hopping is the addition of hops to the wort on the cold side of the brewing process. Unlike standard hop additions, its main purpose is not to produce a characteristic bitterness but to extract as much of the hop essential oils as possible, which are largely lost in the standard hopping process. When dry-hopped, it is possible to obtain a beer with an aroma that is difficult to achieve when hops are used on the hot side of the brewing process. As a result, this process has become very popular in recent years, particularly in beers that belong to the ‘craft beer revolution’ trend. In addition, the usefulness of this process is increasing with the development of new hop varieties with unique aromas. This article presents the main components of hops, focusing on those extracted during the process. Changes in the composition of beer bittering compounds and essential oils resulting from this process are discussed. This paper presents the current state of the knowledge on the factors affecting the degree of extraction, such as hop dosage, the time, and temperature of the process. Issues such as process-related physicochemical changes, hop creep, low flavor stability, haze formation, and green flavor are also discussed.
Введение. В данном обзоре проанализированы современные исследования (с 2015 по 2022 гг.), посвященные вопросу создания ароматического профиля пивоваренной продукции при условии соблюдения всех требований, предъявляемых к готовой продукции в рамках действующих в отрасли стандартов качества.Цель. Целью обзора являлся анализ влияния применяемого растительного сырья, а также микроорганизмов на формирование ароматического профиля пива, в отношении положительных и отрицательных моментов с точки зрения качества готовой пивоваренной продукции. Материалы и методы. Для обзора использованы научные публикации российских и за¬рубежных авторов по вопросам формирования ароматического и вкусового профила пивоваренной продукции за счет органических соединений зернового и растительного сырья. Поиск научных материалов (опубликованных статей) по исследуемой теме на русском и английском языках осуществлялся в базах данных Wed of Science Core Collection, Scopus и eLibrary.ru. В качестве метода исследования использовано обобщение результатов.Результаты. Рассмотрен ограниченный перечень зернового немодифицированного сырья, вклад его органических соединений в ароматический профиль пива. Отмечено положительное влияние разнообразных классов соединений, влияющих на образование летучего профиля пива, с одной стороны, и недостаток низкомолекулярных соединений, обеспечивающих активную бродильную активность микроорганизмов. Рассмотрено влияние специфических органических соединений модифицированного зернового сырья, их вклад в формирование различных ароматических оттенков, что расширяет ассортимент пивоваренной продукции. Рассмотрены нежелательное влияние недостатка низкомолекулярных углеводных и азотистых соединений, а также наличие свободных фенольных форм веществ, способных оказывать негативное влияние на качество пива. Приводится информация о влиянии штаммов дрожжей, их разнообразном метаболизме и вкладе в формирование профиля пивоваренной продукции. Отмечен исследовательский интерес к расширению перечня гибридных штаммов для создания новых органолептических оттенков, которые могут иметь нежелательный характер и привносить в пиво посторонние тона. Выводы. Авторами отмечается, что в рамках действующих требований в рамках ГОСТ 31711-2012, ограничивающих перечень применяемого растительного сырья для производства пива, вопрос формирования различных ароматов все еще остается открытым, несмотря на ощутимые научные достижения в области разнообразия сырьевой базы.
At present, non-alcoholic (NAB) and low-alcoholic beers (LAB) exhibit major staling defects even when fresh, partly due to absence of ethanol as an antioxidant. In the present work, the aroma stability of eleven commercial NABLABs available on the Belgian market, issued from different technological processes, was assessed. NABLABs were investigated, both when fresh and after one year of storage at 20 °C in the dark. Six stale-odorant compounds were found above their perception threshold in aged NABLABs: sotolon, abhexon, methional, phenylacetaldehyde, dimethyltrisulfide and β-damascenone. Based on the chemical structure of the first four, it can be concluded that oxidation is the main issue for NABLABs aging. Yet, five of these usual staling defects of a six-month lager beer were already key-odorants in fresh NABLABs: dimethyltrisulfide, methional, and β-damascenone were above the thresholds in all samples, phenylacetaldehyde in 10 out of 11, and sotolon in 7. In conclusion, development of efficient antioxidants is needed to improve NABLABs acceptability.
Turbidity in beer will continue to be a major topic as many origins of haze have already been discovered, but it is not always possible to identify its immediate cause. In addition to beer foam, one of the most important visual quality characteristics of filtered beers is gloss fineness. Consequently, in the case of an undesirable haze, it is first necessary to identify the haze particles and how to remove them. The topic is covered in two sections. This review labelled Part 1 and a second review labelled Part 2. Part 1 addresses turbidity in general, the causes and origins of turbidity and the different analytical methods for turbidity identification and removal. Part 2 focuses on Raman spectroscopy and provides a general overview of the physical basics, the areas of application, possible gaps and the challenge for its use for identifying turbidity in beer and beverages.
Some polyphenol substances are important for beer production as well as for the health benefits of beer consumption. Hop polyphenols have been relatively well researched in the last decade, less attention has been paid to malt. Our study was aimed to determine the influence of the barley variety and growing locality on the profile of proanthocyanidins (PAC), monomeric flavanols, flavonols and their glycosides in malt. Laboratory malts from three barley cultivars grown in seven countries of the European Union were analyzed by the developed LC-HR / MS method with sample preparation by the QuEChERS technique. Dimers of (epi)catechin (37.0%) and dimers (epi)catechin – (epi) gallocatechin (36.4%) dominate the PAC profile of malt, monomer content being low. Flavanols (epi)catechin and (epi)catechin-O-glucoside have been identified and quantified in malts. The flavonols were quercetin and myricetin, their glycosides were not detected. The results suggest varietal dependence of the PAC, monomeric flavanols and flavonols profile and the lesser impact of the growing locality.
This paper reports the effects of dry-hopping at 4 and 19 °C for a low alpha versus high alpha hop variety on the resulting profiles of non-volatile hop acids (humulinones, iso-α-acids, α-acids). In a dry-hopping study conducted over 2 weeks, we found a significant increase in humulinone concentration driven principally by hop alpha acid content and the duration of dry-hopping. Conclusive evidence of iso-α-acid losses during dry-hopping (by adsorption onto spent hops) is presented, in addition to a significant increase in α-acid concentrations, which was observed only for beers dry-hopped at 19 °C with the high alpha hop variety. Measured beer parameters (especially at 19 °C) revealed an increase in pH, ABV (%), and a decrease in beer density during dry-hopping-from which we conclude that further attenuation of beer occurred during dry-hopping. The polyphenol content of beers was found to increase substantially with dry-hopping time, whilst both temperature and hop variety were found to be significant factors determining the amounts of polyphenols extracted. Finally, analysis of the spent hop slurry (recovered after 14 days of dry-hopping) confirmed that the residual content of hop acids (α-acids, their oxidised derivatives and polyphenol content), makes these materials-currently treated as waste-of potential value for re-use in the brewing process.
Beer composition changes through storage, altering the quality of the product. In the past decade, many papers have been devoted to the compounds responsible for aged-beer off-flavors, mainly trans-2-nonenal, methional, and dimethyltrisulfide. Due to their huge antioxidant activity, polyphenols have often been described as key compounds for limiting beer staling. Yet phenolic structures also evolve through storage. Low-molecular-weight phenols like 4-vinylsyringol can impart off-flavors in aged beer, whilst flavonoids strongly influence astringency, haze, and color. The instability of stilbenes, prenylchalcones, and derived flavanones could also modify their health potential through storage. After reviewing the structures and properties of all phenolic compounds found in beer, this paper will try to assess the impact of their degradation through aging. Extraction procedures for their quantification and treatments for their removal are also described.
Normal-phase high-performance liquid chromatography electrospray ioni-zation(–) tandem mass spectrometry (NP-HPLC-ESI(–)-MS/MS) and thio-acidolysis analyses recently have revealed the presence of (+)-catechin, (–)-epicatechin, procyanidin dimers, and prodelphinidin trimers in acetone/ water (70/30, vol/vol) LH-20 extracts of lager beers. In this study, de-tailed structures were determined by reversed-phase (RP) HPLC-ESI(–)-MS/MS. Four dimers were identified: three procyanidins (B1, B3, and B4) and one prodelphinidin (B3). Previously detected in hop or malt, three trimers (the procyanidin C-4α-8-C-4α-8-C and two prodelphinidins, GC-4α-8-C-4α-8-C and GC-4α-8-GC-4α-8-C) were distinguished for the first time in beer. As expected based on previous thioacidolysis data, most beer proanthocyanidins carried a catechin as the terminal unit.
This study aimed to evaluate the influence of using four different Saccharomyces cerevisiae strains on the phenolic compounds, antioxidant capacity (AOX), organic acid, sugar, and alcohol profiles of American Pale Ale (APA) craft beer. US-05 yeast presented a lower fermentation capacity but originated a beer with suitable ethanol content, higher lactic acid content, and lower acetic acid and succinic acid contents. Furthermore, it presented higher quantities of catechins and procyanidin B1, which resulted in a higher AOX. M15 yeast originated a beer with lower ethanol content and AOX and higher malic and formic acid contents. Beers made with BRY-97 yeast had higher ethanol content, while those made with S-04 yeast presented higher glycerol content. Several factors can affect beer quality. This study demonstrated that the type of yeast strain used in the APA craft beer brewing has a significant impact not only on the fermentative process but also on the functional characteristics of the products.
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.
Multi-stage dry-hopping is a technique that is widely used by brewers to achieve intense hop aroma in beer. However, a thorough analysis of its efficacy does not exist in the published literature. The goal of this study was to compare the properties of beers produced by single or two-stage dry-hopping at the same cumulative rate. On the pilot scale (80-100 L), an un-hopped base beer was subjected alternately to single-or two-stage dry-hop additions at 386, 772, and 1544 g/ hL using Centennial hops. Separately commercial beers ($350 hL batch size) were dry-hopped at 733 g/hL in single or two-stage additions using a combination of Amarillo, Simcoe, Cascade, Citra, and CTZ pellets. Pilot scale dry-hopped beers showed increases in residual extract, pH, bitterness units, humulinones, and total polyphenols, accompanied by a decrease in iso-a-acids with increasing hop dose. Changes in bitterness units, humulinones, and iso-a-acids all appeared to be more pronounced in the two-stage dry-hopped beers. In contrast, commercial beers were nearly identical in terms of chemical composition. Significant increases in six aroma attributes scaled by a trained panel were observed as a result of two-stage dry-hopping in the pilot scale treatments. Slight increases in aroma potential were observed for the commercial beer made with two-stage additions, although these differences were not statistically significant. These results illustrate the impact multi-stage dry-hop additions have on beer aroma, and provide direction to brewers to consider whether multi-stage dry-hopping may yield desirable results while potentially using less hop material.
It has been well established that beer is an excellent source of polyphenols, due in part to the contribution of xanthohumol and other compounds from hops. The intent of this study was to explore the relationship between alpha acid content, antioxidant activity, and boil time in hops. Ten hops strains with different alpha acid contents were analyzed for phenolic antioxidant content via Total Phenolic Concentration (TPC) and Ferric Reducing Antioxidant Power (FRAP) assays. Each strain was boiled for 10, 30, or 60 min, consistent with typical beer brewing protocols. Phenolic and antioxidant content increased with boil time, with Citra hops showing higher phenolic content than other strains regardless of boil time. It appears that longer boil times yield higher TPC and FRAP concentrations regardless of alpha acid content. There was a nonstatistical inverse correlation between alpha acid and phenolic content; as a result, beers that use high alpha acid hops or short boil times may have lower TPC and FRAP concentration than beers that use low alpha acid hops and long boil times.
After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. As free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21), and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by NMR and HRMS. HPLC-ESI(+)-MS/MS evidenced both for the first time in hop. Both S-conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Like G-23, G-21 appeared ubiquitous to all varieties. Of all the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg per kg of dry matter. Cys-21 was found in all samples at much lower concentration (up to 0.2 mg per kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed to confirm that brewing yeast is able to release free 21 from them.
Enzymes in hops have recently been demonstrated to hydrolyze beer dextrins, produce fermentable sugars in finished beer, and pose significant safety and quality challenges for brewers. In an effort to mitigate beer refermentation following dry-hopping in the presence of yeast and the effect of "hop creep," brewers look to adjust recipes, selection of ingredients, and dry-hopping conditions. With phytochemical/secondary metabolite based flavor differences known to exist across hop cultivars, it was posited that there may also exist unique enzymatic activities based upon cultivar and that brewers may use these differences to guide their hopping practices. The enzymatic power of 30 hop cultivars was screened using specific enzyme assay kits (a-amylase, b-amylase, amyloglucosidase) on hops as well as quantifying via liquid chromatography the non-fermentable dextrin degradation and sugar production in beer dry-hopped with different hops. Differences across all cultivars were found and cluster analysis revealed groupings that were not based on pedigree, genetic makeup, or specific enzyme activities. The influence of growing and harvest practices, processing conditions, and presumably age post processing on hop enzymatic power warrants more investigation. These findings indicate that the choice of hop cultivar may influence the degree of refermentation in finished beer as a result of dry-hopping.
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 ability of tannins to self-associate or form complexes with other macromolecules has important nutritional
implications but can also result in defects in beverages. In addition, oxidation may be involved in the aggregation
properties of tannins.
In order to assess the impact of tannin oxidation on their self-association, oligomeric procyanidins were
oxidized in a model solution and their aggregation kinetics were studied using light scattering.
Under the conditions tested, only oxidized procyanidins were involved in haze formation. An increase in the
level of oxidation and the degree of polymerization of procyanidins enhanced aggregation.
Procyanidin oxidation products were depolymerized and the evolution of their markers was monitored
throughout the aggregation process using liquid chromatography coupled with mass spectrometry. This revealed
the involvement of intramolecular coupling in reversible haze formation.
The haze formed in a model solution was partially reversible at high temperature. This property was similar in
pommeau, an apple-based beverage. This work highlighted the involvement of oxidized tannins in reversible
haze.
The range of different nonvolatile constituents extracted from hops in highly hopped beers suggests that isohumulones may not be the sole contributor to beers' bitterness. Among brewers producing hop-forward beer styles there is concern that the Bitterness Unit (BU) is no longer an accurate predictor of beer bitterness. This study examined factors within the beer matrix that influence sensory bitterness perception in highly hopped beers. Over 120 commercial beers were evaluated using sensory and instrumental techniques. Chemical analysis consisted of the BU via spectrophotometry, hop acids via HPLC, total polyphenols via spectrophotometry, and alcohol content plus real extract via an Alcolyzer. Sensory analysis was conducted over two studies, and the beers' overall bitterness intensity were rated using a 0-20 scale. This study identified that the BU measurement predicts sensory bitterness with a nonlinear response, and it proposed an alternative approach to predicting bitterness based on isohumulones, humulinones, and ethanol concentrations. The study also revealed the importance of oxidized hop acids, humulinones, as a significant contributor to beer bitterness intensity.
The impact on analytical and sensory bitterness of hop acids and hop-derived polyphenols resulting from dry-hopping in beer was investigated using a pilot-scale dry-hopping study and a commercial dry-hop survey. The pilot-scale dry-hopping study utilized a trained sensory panel to quantify increases in bitterness caused by dry-hopping an unhopped ale at different dosing rates (0–16 g/L) and exposure times (0–72 h). The International Bitterness Unit (IBU) and a range of hop acids and polyphenols were measured in the dry-hopped beer to determine which specific bitter hop components may have been responsible for dry-hopping bitterness. The commercial survey examined the bitter acids and polyphenol chemistry of 15 different commercial beers, pre- and post-dry-hopped, brewed by Pacific Northwest breweries. Although iso-a-acids (IAA) were the main contributor to beer bitterness, humulinones (oxidized a-acids) and polyphenols were also potentially significant contributors to bitterness, particularly in heavily dry-hopped beer. The increase in beer bitterness (IBU) as a result of dry-hopping was attributed to humulinone extraction and, in some cases, polyphenol extraction. The commercial survey noted a decrease in the total IAA concentrations as a result of dry-hopping in a majority of the samples tested, indicating that the dry-hopping process may remove IAA from beer.
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.
Thirty-four commercial lager beers were analysed for their hop bitter acid, phenolic acid and polyphenol contents. Based on analytical data, it was evident that the beers had been produced using a range of different raw materials and hopping practices. Principal Components Analysis was used to select a sub-set of 10 beers that contained diverse concentrations of the analysed bitter compounds. These beers were appraised sensorially to determine the impacts of varying hop acid and polyphenolic profiles on perceived bitterness character. Beers high in polyphenol and hop acid contents were perceived as having ‘harsh’ and ‘progressive’ bitterness, whilst beers that had evidently been conventionally hopped were ‘sharp’ and ‘instant’ in their bitterness. Beers containing light-stable hop products (tetrahydro-iso-α-acids) were perceived as ‘diminishing’, ‘rounded’ and ‘acidic’ in bitterness. The hopping strategy adopted by brewers impacts on the nature, temporal profile and intensity of bitterness perception in beer.
Liquid chromatography–tandem mass spectrometry (LC/MSn) coupled with various two-dimensional NMR methods are among the most practical approaches for analyzing proanthocyanidins from hops. Using these and other analytical approaches, it has been shown that hops contain a preponderance of polymeric material, including proanthocyanidins which are dominant, but small quantities of prodelphinidins and propelargonidins are also present. The flavanoids (+) catechin and proanthocyanidin B3 are the most representative followed by B1, B4 and C2. The relative proportions of the various proanthocyanidins vary greatly depending on the variety and climatic conditions as shown by differences in the proanthocyanidins in the same cultivar from different geographic regions. Proanthocyanidins have received a considerable amount of attention in the brewing industry because of their tendency to complex with proteins and contribute to non-biological haze formation, but they stabilize the organoleptic properties and color, and contribute to the astringency and bitterness, which are desirable attributes. Recent studies also show that these compounds, which are classic antioxidants, may also have significant beneficial health effects. Numerous proanthocyanidins including catechin, prodelphinidin B3, procyanidin B3 and epicatechin are present in beer.
A radio-dilution technique has been developed for estimating levels of dimeric polyphenols in barley, malt, wort, and beer. Levels present in a number of different cultivars of barley vary from 130 to 350 mg/kg. When barley is malted, the loss of dimeric polyphenols is normally less than 15%. However, large losses of dimers occur during mashing and wort boiling, and smaller losses usually take place during fermentation and conditioning. The higher the level of dimeric polyphenols in the malt, the higher the level in the derived beer.
The rate of oxidation of SO2 was studied in a wine-model system under aerial oxygen saturation conditions to gain further insight into its mechanism of action in wine. When SO2 was studied alone, no significant oxidation was observed unless iron and copper were introduced. When these metals were added a slow oxidation was observed and bound SO2 also increased to a small but significant extent. These results are consistent with a radical chain mechanism initiated by metal catalysis, in which powerful oxidizing radicals, capable of oxidizing ethanol to acetaldehyde, are produced. This increase in bound SO2 is prevented by 4-methylcatechol (4-MeC) in keeping with the known ability of polyphenols to scavenge these intermediate radicals and thus to inhibit SO2 autoxidation, which consequently should not occur in wine. When 4-MeC was introduced at a concentration that simulated the reducing capacity of red wine, again no significant SO2 oxidation was observed without addition of iron and copper. Had the catechol been oxidized, hydrogen peroxide would have been generated and reacted with the SO2. In the presence of both metals the rate of SO2 oxidation was markedly increased compared to SO2 alone and then was dependent on the concentration of the catechol. These results demonstrate the crucial importance of metals in allowing polyphenol oxidation and that the rate of SO2 consumption is dependent on the rate of catechol oxidation. When iron and copper were added separately, only a modest increase in rate of catechol oxidation resulted. However, when combined, marked synergism was observed and the rate then became very sensitive to copper concentration. It is proposed that copper, by interacting with oxygen, facilitates redox cycling of iron. Exposure of a red wine to the conditions used in this study produced similar results regarding SO2 oxidation to those observed. Copyright © 2007 by the American Society for Enology and Viticulture. All rights reserved.
Several substances can cause haze in beer, but the most frequently encountered problem is due to a cross-linking of polyphenol and protein. These materials probably exist in equilibrium in beer and manifest themselves as a haze when the polyphenol polymerizes. The proteins particularly involved in haze are rich in proline. A range of stabilization treatments - of relatively low cost - is available, but the avoidance of haze problems (which seem to be occurring with increased frequency) demands a holistic approach from the brewer in which all stages from raw material selection to final processing are geared to achieving prolonged shelf life.
Despite the key role of flavan-3-ols in many foods, very little is yet known concerning the modification of their chemical structures through food processes. Degradation of model media containing (-)-epicatechin and procyanidin B2, either separately or together, was monitored by RP-HPLC-DAD-ESI(-)-MS/MS. Medium composition (aqueous or lipidic) and temperature (60 and 90 °C) were studied. In aqueous medium at 60 °C, (-)-epicatechin was mainly epimerized to (-)-catechin, but it was also oxidized to "chemical" dimers, a "chemical" trimer, and dehydrodi(epi)catechin A. Unlike oxidation, epimerization was enhanced at 90 °C. In lipidic medium, epimerization proved slow but degradation was faster. Procyanidin B2 likewise proved able to epimerize, especially at 90 °C and in aqueous medium. At high temperature only, the interflavan linkage was cleaved, yielding the same compounds as those found in the monomer-containing model medium. Oxidation to procyanidin A2 was also evidenced. With little epimerization and slow oxidation even at 90 °C, procyanidin B2 proved more stable in lipidic medium. Synergy was also observed: in the presence of the monomer, the dimer degradation rate increased 2-fold at 60 °C. This work states for the first time the presence of newly formed flavan-3-ol oligomers in processed cocoa.
Different concentrations of catechins and procyanidins were added to an all-malt Pilsner beer brewed from proanthocyanidin-free malt (ant 13·13 × Rupal) and tannin-free hop extract in order to evaluate their intrinsic role in beer haze formation, i.e. in the absence of malt and/or hop flavanoids. The molar tanning capacities of flavanoids depend upon their degree of polymerisation. Procyanidin B6 was much more haze active than procyanidin B3. Both immediate haze and formation of haze after 150 days of storage at room temperature was in general linearily related to the added concentration of phenolics.
Although hop technology has been a substantial part of brewing science for the last 130 years, we are still far from claiming to know everything about hops. As hops are considered primarily as a flavour ingredient for beer, with the added benefit of having anti-microbial effects, hop research is focused on hops as a bittering agent, as an aroma contributor and as a preservative. Newer fields in hop research are directed toward the relevance of hops in flavour stability, brewing process utilisation, the technological benefits of hops in brewing as well as hops as a source of various substances with many health benefits. However the more we find out about the so-called “spirit of beer” the more questions emerge that demand answers. While hop research was only an ancillary research field for decades, during the last ten years more universities and breweries have determined that hops must play a meaningful role in their research efforts. This article gives an overview of the up-to-date knowledge on hop aroma, hop derived bitterness, and the role of hops in flavour stability as well as light stability. Hop research is a wide field, therefore in this review only selected topics are reviewed. Other research areas such as hops utilisation, the antifoam potential of hops, or the advances in knowledge pertaining to the physiological valuable substances of hops go beyond the scope of this article.
Haze formation in beer is claimed to involve the gradual polymerization of polyphenols and their subsequent reaction with proteins to form insoluble complexes. The mechanism of haze formation has now been studied using 14C-labelled epicatechin and dimeric catechin. Epicatechin does not polymerize to form dimeric or trimeric polyphenols when beer is stored and is only incorporated into beer haze to a small extent. Some of the epicatechin combines with nitrogen-containing compounds during storage to form soluble complexes. However, dimeric catechin does not form soluble complexes with nitrogen-containing compounds and instead there is a substantial incorporation of this dimer into beer haze. Dimeric polyphenols are important haze precursors which form insoluble complexes with one or more of the polypeptides of beer, probably by an oxidative coupling mechanism.
This work describes a method for the quantitative analysis of flavanoids of barley, hops and beer by HPLC. This method requires only about 4 h and it gives representative and reproducible results. A few results obtained by this method are given for barley and hops.
The composition of flavonoids in plants is thought to be genetically determined. Thus it is characteristic for plant species. Here, proanthocyanidins are used to identify barley (Hordeum vulgare L.) varieties: six barley varieties and the corresponding malts in 61 samples of the growing years 1998–2001 from three locations in Germany are examined. Their absolute polyphenolic composition is quite similar, but the ratios of proanthocyanidins among each other—termed here “relative quantitative fingerprint” (RQF)—are characteristic for the variety independent from growing year, provenience and status (barley/malt). The analytical method presented is highly automated and robust, thus it allows analyzing a high number of samples and to detect small differences between samples unambiguously. Hence the polyphenols can be used as a tool for food tracing, to prove authenticity and to discover frauds of food from plant origin.
The rates of epimerization of (+)-catechin (1) to (+)-epicatechin (2) and of (-)-epicatechin to (-)-catechin in aqueous solution were measured over the pH range 5.4-11.0 and the temperature range 34-100°C. The rate of conversion of (+)-catechin to catechinic acid (3) also was measured under these conditions. First-order kinetics were observed for all three processes. At low pH, k(epimerization) ≫ k(rearrangement), and epimerization approached an equilibrium in which (+)-catechin predominated over (+)-epicatechin. Near pH 11 and at elevated temperatures, k(epimerization) was only slightly greater than k(rearrangement), and the rapid, irreversible formation of catechinic acid under these conditions determined product composition. Both the epimerization of catechin and its rearrangement to catechinic acid can be rationalized in terms of a quinone methide intermediate (4).
A semipreparative chromatographic method for the isolation of small amounts (10−20 μg) of dimeric and trimeric proanthocyanidins from barley is described. Concentrated extracts of barley were injected onto a high-performance gel filtration column (Superdex 75 HR), and were eluted with methanol. This procedure resolved the dimeric proanthocyanidins (prodelphinidin B3 and procyanidin B3), as well as the trimeric procyanidin C2 and three other trimeric prodelphinidins. The separated flavanoid peaks were collected and their contents were estimated by UV spectrophotometry, reaction with p-dimethylaminocinnamadehyde, and reversed phase HPLC with electrochemical detection. This method produced proanthocyanidins in sufficient amounts to calibrate a system for direct injection chromatographic analysis of beers and barley extracts. The method described may be optimized for the isolation of dimeric proanthocyanidins only, in which case the preparation can take as little as 3 h; alternatively, by extending the chromatographic separation to 9 h, the four major trimeric proanthocyanidins of barley can be recovered also in a chromatographically pure state. Keywords: Analysis; barley; beer; calibration standard; high-performance liquid chromatography; proanthocyanidin
Gradient elution from a reversed-phase high-performance liquid chromatography column was used to separate and quantitate the flavonol glycosides and simple flavanol oligomers extracted from hops and barley. Whereas flavonol monoglycosides, diglycosides and triglycosides, and flavanol monomers, dimers and trimers were readily separated, polymeric flavanols were not resolved. A column temperature of 30°C was found optimal for the separation of flavonol aglycones by isocratic elution.
J. Inst. Brew. 116(4), 369–380, 2010 Generally referred to as polyphenols (PPs), beer flavonoids such as the flavan-3-ols and their condensed products, the proantho-cyanidins, represent a class of readily oxidizable compounds capable of hindering or preventing the oxidation of other mole-cules present in beer. Flavan-3-ol and proanthocyanidin capacity to improve oxidative stability has been well established in other food systems, and thus these antioxidants have recently gained significant consideration as potential beer flavour modifiers and/or stabilizers. The duality of their presence in beer is that PPs complex with proteins in the beer matrix to form temporary and permanent hazes. Undesirable physical instability caused by PP-protein interactions can be resolved via use of adsorptive resins such as polyvinylpyrrolidine. While there is no doubt that polyphenol removal increases beer shelf stability in terms of haze formation, the impact of polyphenol removal on beer fla-vour remains unresolved. This review discusses the sources, content and impact of polyphenol presence and removal on beer physical and flavour stability.
(2R,3S)-Catechin-7-O-#-D-glucopyranoside is isolated from barley (Hordeum vulgare L.) and malt for the first time. For identification, the glucoside was isolated from acetone-water extracts of malt by polyamide clean-up and semi-preparative HPLC. The structure was elucidated by various NMR techniques, fast atom bombardment mass spectrometry and UV and circular dichroism spectroscopy. The investigation of 20 different barley varieties and the corresponding malts by HPLC with UV and coulometric electrode array detection revealed that the amount of the glucoside increases during malting. In contrast, the content of the monomeric (+)-catechin decreases and (-)-epicatechin was not detected.
Since lipid auto-oxidation during wort boiling is a determining for the appearance of staling flavour in aged beers, we have investigated the reducing power of hops added in the boiling kettle. An assay based on the inhibition of linoleic acid oxidation in the presence of an initiator [2,2′-azobis(2-amidino-propane) dihydrochloride=AAPH] enabled us to distinguish hop varieties and conditionings. Large differences in hop flavanoid contents explained the higher antioxidant activity of low-α-acid samples versus bitter varieties and CO2 hop extracts. As expected, adding hop pellets to the kettle effectively increased the overall reducing activity of wort. Supercritical CO2 hop extracts had no significant effect due to their extremely low level of polyphenols. The concentration of the very well-known marker of beer ageing, trans-2-nonenal, was lower in boiled wort exhibiting a better reducing power. The AAPH reducing power test applied to hops or worts was thus efficient to predict the nonenal synthesis during boiling. Hop varieties and conditionings emerged from this work as key-parameters for improving the reducing power of wort and the flavour stability of the final product.
Proanthocyanidins are well known for their involvement in haze and colour development during beer ageing. New methodologies are needed, however, to understand what happens to them in the bottled beer. For the first time in the brewing field, thiolysis was hyphenated to RP-HPLC-ESI(-)-MS/MS to investigate these flavanoids. Thirty minutes at 40°C followed by 10h at room temperature emerged as the best conditions for complete depolymerisation. NP-HPLC-ESI(-)-MS/MS was used to quantify and isolate fractions from monomers to trimers in a Sephadex LH20 acetone/water (70/30, v/v) beer extract. Unsurprisingly, a lower dimer/monomer ratio was evidenced in PVPP-treated beers than in silica gel-filtered beers. Most beer dimers are procyanidins B3 (two catechin units) whilst most trimers are prodelphinidins (catechin in terminal units and gallocatechins or catechins in extension units). Gallocatechin appeared to come mainly from malt. Despite the absence of chromatographic peaks corresponding to oligomers above trimers, an apparent degree of polymerisation close to six was calculated in the total LH20 extract. Still higher mean degrees of polymerisation (mDPs) were calculated for malt and hop, indicating selective extraction or depolymerisation from raw materials to beer. The main part of beer polyphenols is composed of complex undefined structures not degraded by toluene-α-thiol.
Copyright © 2008 Elsevier Ltd. All rights reserved.
Procyanidins (i.e. condensed tannins) are polyphenols commonly found in fruits. During juice and cider making, apple polyphenol oxidase catalyzes the oxidation of caffeoylquinic acid (CQA) into its corresponding o-quinone which further reacts with procyanidins and other polyphenols, leading to the formation of numerous oxidation products. However, the structure and the reaction pathways of these neoformed phenolic compounds are still largely unknown. Experiments were carried out on a model system to gain insights into the chemical processes occurring during the initial steps of fruit processing. Procyanidin B2 was oxidized by caffeoylquinic acid o-quinone (CQAoq) in an apple juice model solution. The reaction products were monitored using high performance liquid chromatography (HPLC) coupled to ultraviolet (UV)-visible and electrospray tandem mass spectrometry (ESI-MS/MS) in the negative mode. Oxidative conversion of procyanidin B2 ([M-H](-) at m/z 577) into procyanidin A2 at m/z 575 was unambiguously confirmed. In addition, several classes of products were characterized by their deprotonated molecules ([M-H](-)) and their MS/MS fragmentation patterns: hetero-dimers (m/z 929) and homo-dimers (m/z 1153 and 705) resulting from dimerization involving procyanidin and CQA molecules; intramolecular addition products at m/z 575, 573, 927, 1151 and 703. Interestingly, no extensive polymerization was observed. Analysis of a cider apple juice enabled comparison with the results obtained on a biosynthetic model solution. However, procyanidin A2 did not accumulate but seemed to be an intermediate in the formation of an end-product at m/z 573 for which two structural hypotheses are given. These structural modifications of native polyphenols as a consequence of oxidation probably have an impact on the organoleptic and nutritional properties of apple juices and other apple-derived foods.
A method was developed to determine the flavanols prodelphinidin B3, procyanidin B3, (+)-catechin, and (-)-epicatechin by high-performance liquid chromatography, using dual-channel electrochemical detection. This method was especially suited to the direct analysis of beer samples and to analysis of acetone extracts of barley samples, and was capable of determining proanthocyanidins and catechins at levels of 0.1-5.0 mg l-1. The use of dual-channel electrochemical detection also enabled unambiguous peak identification by measurement of collection efficiencies. This method offered improved sensitivity and selectivity compared with ultraviolet detection, and sample preparation procedures were greatly simplified. The method was applied to the comparison of stabilized and unstabilized lagers, and to the analysis of different barley varieties grown in Ireland.
A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.
The use of labeled nonenal enabled the demonstration that the appearance of the cardboard flavor in finished beer comes from lipid auto-oxidation during wort boiling and not from lipoxygenasic activity during mashing. Free trans-2-nonenal produced by linoleic acid auto-oxidation in the kettle disappears, owing to retention by wort amino acids and proteins. This binding linkage protects trans-2-nonenal from yeast reduction but is reversible, allowing release of the compound at lower pH during aging. Labeled trans-2-nonenal is detected after aging when deuterated precursors form in the boiling kettle. The amount of alkenal released correlates with the concentration of reversible associations in the pitching wort. This work brings new illumination to the formation of trans-2-nonenal and overturns many previous hypotheses. It also explains why a reduction in the beer pH intensifies the cardboard flavor.
Of three different solvents (acetone, ethanol, and methanol) mixed with water and acetic acid, the acetone/water/acetic acid mixture (70:28:2, v/v) proved to be best for extracting dark-chocolate procyanidins. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS-ESI) was further used to identify oligomers found in the extract. After HPLC fraction collection, the reduction power of flavanoid fractions was measured in the AAPH [2,2'-azobis(2-amidinopropane)dihydrochloride] assay, where oxidation of linoleic acid is induced in an aqueous dispersion. Even expressed in relative monomeric efficiency units, the oxidation-inhibiting power of polymerized oligomers is much stronger than that of monomers. A comparison with 10 usual antioxidants indicated that oligomers with three or more (epi)catechin units are by far the most efficient.
An analytical method based on solid-phase extraction (SPE) and followed by liquid chromatographic separation and ultraviolet detection (HPLC-UV) is proposed for the determination of 10 phenolic compounds which participate on beer stability and sensory properties in alcohol-free beers. Acetonitrile was found to be the most appropriate solvent for the elution of polyphenolic compounds adsorbed on C18 cartridges. The performance of the method was assessed by the evaluation of parameters such as absolute recovery (generally higher than 60%), repeatability (lower than 10%), linearity (r2 higher than 0.993) and limits of quantitation (ranging from 1 to 37 microg/L); no matrix effects were observed. The polyphenol content of different Spanish alcohol-free beers is presented. Five phenolic compounds such as protocatechuic, p-coumaric, ferulic, caffeic acids, and (+)-catechin were identified at levels lower than 10 mg/L.
Besides Maillard reactions, structural rearrangements of flavan-3-ol monomers cause color changes in beer during storage. Acetone/water-soluble fractions (70/30, v/v) of three lager beers of the same batch, differently stabilized before bottling in glass or poly(ethylene terephthalate) (PET) bottles, were monitored by normal-phase HPLC-ESI(-)-MS/MS over a 1-year period of storage at 20 degrees C. In parallel, beer color was monitored by the European Brewery Convention assay. The evolution of color was similar in the silica gel-filtered beer to that in identically bottled and stored poly(vinylpolypyrrolidone)-treated samples, despite the high flavanoid dimers content of the former. On the other hand, color evolved more rapidly in the PET bottle, suggesting a key role of oxygen. The kinetics was still increased in model media containing (+)-catechin, while no color was detected when normal-phase HPLC-fractionated dimers or trimers were investigated. (+)-Catechin emerged as the precursor of less polar products, characterized by a yellow-brown color. MS/MS enabled us to identify these products as issued from the oxidation and intramolecular additions of dehydrodicatechin B4. Similar structures were found in aged beers spiked with (+)-catechin. Beer storage in the absence of oxygen and at low temperature is recommended so as to minimize the synthesis of such pigments.
The pattern of the monomeric and oligomeric flavan-3-ols for 10 barley varieties and the corresponding malts were identified and quantified using high-performance liquid chromatography-ultraviolet detection-electrospray ion trap mass spectrometry. The Folin-Ciocalteau and the vanillin spectrophotometric assays were used for the assessment of the total polyphenol and total flavan-3-ol content, respectively, and the antioxidant activity was determined as the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and the ferricyanide reducing power. Catechin and prodelphinidin B3 were respectively the major monomeric and dimeric flavan-3-ols. Moreover, prodelphinidin B3 was shown to be the main contributor for the radical scavenging activity both for barley and malt.
Humulinone Formation in Hops and Hop Pellets and Its Implications for Dry Hopped Beers
- J P Maye
- R Smith
- J Leker
Quantification Methods, and Stability of Phenolic Compounds in Beer - A
- D Callemien
- S Collin
- Organoleptic Structure
- Properties
The Use of 14 C-Labelled Dimeric Catechin to Study the Fate of Dimeric Polyphenols during Malting and Brewing
- D R J Laws
- J D Mcguinness
- N A Bath