Production of novel varietal hop aromas by supercritical fluid extraction of hop pellets—Part 1: Preparation of single variety total hop essential oils and polar hop essences

J. of Supercritical Fluids 69 (2012) 45–56

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Production of novel varietal hop aromas by supercritical fluid extraction of hop pellets—Part 1: Preparation of single variety total hop essential oils and polar hop essences Filip Van Opstaele ∗ , Koen Goiris, Gert De Rouck, Guido Aerts, Luc De Cooman KAHO Sint-Lieven, Association KU Leuven, Department of Microbial and Molecular Systems (M2S), Leuven Food Science and Nutrition Research Centre (LFoRCe), Laboratory of Enzyme, Fermentation and Brewing Technology, Technology Campus, Gebroeders De Smetstaat 1, 9000 Gent, Belgium

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Article history: Received 20 February 2012 Received in revised form 8 May 2012 Accepted 23 May 2012 Keywords: Supercritical carbon dioxide Hop essential oil Hop aroma Beer aromatisation Sensory evaluation Flavour thresholds

a b s t r a c t A new methodology for the selective isolation of varietal total hop essential oil from hop pellets and further chromatographic fractionation of total hop oil into terpeneless polar hop essence, is presented. The methodology is essentially based on supercritical fluid extraction (SFE) of total hop essential oil using carbon dioxide of appropriate density, followed by solid phase extraction (SPE) using octadecylsilica and ethanol/water mixtures for preparation of polar hop essence. Different SFE temperature–pressure combinations were tested for extraction of total hop essential oil from pellets. A carbon dioxide density of 0.50 g/mL (50 ◦ C, 110 atm), proved to be the best compromise in view of selective and quantitative extraction of total hop essential oils. Further fractionation of total hop essential oil by SPE in order to remove hydrocarbons, resulted in varietal polar hop essence, highly enriched in water-soluble, oxygenated hop oil constituents. All of the applied procedures of the proposed SFE/SPE methodology are in full accordance with the principles of clean-label technology. As a result, the novel hop aroma products are fully compatible with the beer matrix. When added to beer, the novel hop oil preparations impart a typical, varietal dependant pleasant hoppy character and increase beer bitterness and mouthfeel. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Next to the brewing water, malt and yeast, hops (Humulus lupulus L.) constitute a basic raw material for beer production [1]. Although quantitatively hops represent only a minor ingredient, it has an enormous impact on final beer flavour. Indeed, the sensory flavour profile of unhopped beer is mainly dominated by a rather unpleasant malt flavour and too much sweetness and alcoholic flavour [2]. Hops are therefore essential and even unique for beer as typical hop-derived beer bitterness and a more or less pronounced hoppy aroma differentiate beer from any other alcoholic beverage. Several types of secondary metabolites are at the origin of the characteristic flavour attributes hops impart to beer, i.e. the hop acids, particularly the ␣-acids, the components of the essential oil, and the hop polyphenols. Beer bitterness and hoppy aroma are derived from the ␣-acids and the essential oil, respectively, whereas hop polyphenols contribute to mouthfeel and especially fullness of beer [1,3–8].

∗ Corresponding author. Tel.: +32 09 265 86 13; fax: +32 09 265 87 24. E-mail address: fi[email protected] (F. Van Opstaele). 0896-8446/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2012.05.009

Hop-derived beer bitterness is caused by ␣-acids isomerisation during wort boiling into the bitter tasting trans- and cis-iso-␣-acids and thus well-defined from a chemical point of view [9,10]. Moreover, in current brewing practice, beer bitterness can be adequately dosed and controlled because of the availability of advanced hop products with known amounts of pre-isomerised (reduced) iso-␣acids [6,11–14] and optimised analytical methods allowing reliable quantification of beer bittering principles [15,16]. In sharp contrast with the role of hop-derived iso-␣-acids in beer bitterness, the precise nature of hoppy aroma of beer is however far from understood. Although it is generally recognised that mainly volatile constituents present in, or derived from, hop essential oil are involved [1], to date, hoppy aroma is still one of the most controversial aspects of beer flavour as key components responsible for it remain to be identified despite many years of intensive research [17–21]. In conventional brewing practice, typical hop aromatisation technologies such as late hopping at the end of the boil or dry hopping during lagering, using hop cones or pellets of aroma varieties, are applied to impart a desired hoppy aroma and flavour effect. However, these practices may lead to relatively large, unwanted fluctuations in hop character of the final product, due to numerous parameters and variables related to the hops and the brewing process, such as the selected hop variety, the provenance and crop

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year of the hops, post-harvest hop storage and processing conditions, hopping rate(s) and point(s) of addition, time and vigour of wort boiling, and fermentation and maturation conditions [22–36]. Efforts to circumvent drawbacks of conventional hop aromatisation have led to the development of several types of advanced hop oil products, based on knowledge of chemical/sensory aspects of hop essential oils, hopped wort, and beer [6,37–43]. Nowadays, these hop oils or fractions thereof are commercially available and they appear to offer an elegant and versatile tool in view of the production of beers with a more consistent and distinct hoppy aroma [42,43]. According to the EBC Manual of Good Practice ‘Hops and Hop Products’ [6], commercially available hop oil products are classified into oil-rich hop extracts and pure hop oils/hop essences. Total or pure hop oils, comprising several hundreds of volatiles (hydrocarbons, oxygenated compounds, sulfur-containing compounds), are generally isolated from hops in two steps. In the first step, pure resin extracts or oil-rich hop extracts are produced via liquid or supercritical carbon dioxide extraction. Pure resin extracts contain the most important brewing constituents (i.e. hop acids and hop essential oil). Usually, an extraction pressure of 60–65 bar and a temperature of 5–15 ◦ C are applied in the liquid carbon dioxide extraction process, whereas a pressure of 200–250 bar and a temperature of 40–60 ◦ C is used for supercritical extraction with carbon dioxide. Oil-rich hop extracts are enriched in hop essential oil and also still contain some hop acids. Oil-rich hop extracts are obtained via partial extraction of hops with liquid carbon dioxide (0 ◦ C, 60 bar) or supercritical carbon dioxide extraction at relatively low pressure (40–60 ◦ C, up to 120 bar) [6]. Next, pure hop oils are then isolated from the extracts in the second processing step by steam distillation (under vacuum or at atmospheric pressure) or via molecular distillation under high vacuum [6,19]. Pure hop oils are available as either varietal or generic preparations and are usually added post-fermentation (but before final beer filtration), to convey dry hop aroma and flavour. Further fractionation of total hop oils into several types of hop essences is achieved using a combination of distillation and chromatographic methods [19,39,43]. However, precise information on the procedures applied for production of these essences is kept confidential [6,19]. Fractionation of total hop oils results in varietal or generic hop oil essences. Varietal essences represent the water-soluble or oxygenated fraction of total varietal hop essential oil and are used to impart late or dry hop flavour notes. Generic essences are grouped into floral, spicy, herbal, citrusy, estery, and sylvan essences based on the organoleptic impressions they impart when added to beer. Both varietal and generic essences may be added to bright beer to introduce the desired hop-derived flavour impressions [43]. The potential of commercially available hop oil products for beer aromatisation was previously investigated at our institute in close collaboration with several Flemish breweries [41,44,45]. In general, additions of appropriate amounts of total hop essential oils or varietal ‘dry hop’ essences to beers, were clearly appreciated, in particular when added to top-fermented beers. When these hop aromas were used in combination with pre-isomerised hop extracts, the resulting fully advanced hopped beers were even preferred to conventionally pellet hopped reference beers. However, in case of application of generic ‘late hop’ essences (floral and spicy essences), the resulting flavour impressions did not come up to expectations such as a clear floral or spicy hop note, which is probably due to the still relatively complex composition of commercial ‘late hop’ essences [41,45]. Therefore, in this study, we aimed at developing a new technology which, in sharp contrast with industrial protocols, directly and selectively isolates total varietal hop essential oils from hop pellets and further fractionates the isolated hop oils into highly enriched varietal hop essences which impart distinct, pleasant flavour impressions when added to beer, i.e. dry hop character or floral, citrusy, and spicy hop notes.

Supercritical fluid extraction (SFE) of hop pellets T90 with carbon dioxide was chosen as the extraction technique since it completely fits into our rationale of clean-label technology and full compatibility of the hop oil products with the beer matrix. According to Mukhopadhyay [46], supercritical carbon dioxide brings about the most natural odour and taste in the extracts, bearing the closest resemblance to the original material when compared with all other available and allowable solvents for extraction. With regard to the extraction of hops, carbon dioxide proves to be highly efficient for selective isolation of the essential brewing principles from hop resin, i.e. the hop acids and the essential oil [6,12,39,47]. Furthermore, it does not react with the material being extracted, there are no harmful solvent residues left in the final preparation, and it is a natural by-product of fermentation. Interestingly, when using carbon dioxide in the supercritical fluid state, the density and thus the dissolving power may be altered by variation of pressure and temperature, allowing fractionated extraction of total hop oils (low carbon dioxide density) and hop acids (high carbon dioxide density), as previously demonstrated by Verschuere et al. [48]. In this work, the unique properties of supercritical carbon dioxide are evaluated for selective extraction of different types of hop aromas. The focus of Part 1 of our study1 is on selective supercritical fluid extraction (SFE) of single variety total hop oils from pellets and on subsequent fractionation of these pure hop oils into more water-soluble, oxygenated polar hop essences. The following starting-points form the basis of this new methodology: - The hop oils are to be extracted directly from hop pellets instead of starting from oil-rich hop extract or pure resin extract, in order to minimise risks of modification of the original hop oil composition. - Taking into account varietal dependence of hoppy aroma of beer [21,22,49–52], all hop oils and essences will be prepared from single hop varieties and their characteristic varietal origin will be preserved throughout the extraction/fractionation process. - The whole extraction/fractionation process must be in full accordance with the principles of clean-label technology so that indisputable compatibility of all novel hop aromas with the beer matrix is achieved. Therefore, SFE using only carbon dioxide (i.e. no use of organic modifiers) is selected as the extraction technique of choice. - Upon further fractionation of SFE-preparations by solid phase extraction (SPE), only water, ethanol or mixtures thereof, are allowed as eluents, in view of full compatibility of the purified hop oil products with the beer matrix. 2. Materials and methods 2.1. Chemicals The following chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) and were of analytical grade: ␤-caryophyllene (98.5%), caryophyllene oxide (≥99%), ␣-copaene (≥90%), dichloromethane (≥99.9%), ␣-humulene (≥98%), linalool (≥98%), ␤-myrcene (≥95%), nonadecane (99%), 2-undecanone (99%). Calcium chloride dihydrate (≥99.0%) was purchased from Merck (Darmstadt, Germany); Carbon dioxide (≥99.998%) was purchased from Air Liquide Benelux (Luik, Belgium); ethanol absolute (≥99.8) was purchased from VWR International (Zaventem, Belgium); milli-Q water was obtained from a milli-Q purification system (Synergy 185, Millipore S.A., Molsheim, France).

1 Part 2: Preparation of single variety floral, citrus, and spicy hop oil essences by density programmed supercritical fluid extraction.

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Fig. 1. Scheme of the supercritical fluid extractor (Dionex SFE-703) [53].

2.2. Plant material Hop pellets T90 (crop year 2005) from different varieties (cv. Saaz, cv. East Kent Golding, cv. Hersbrucker Spät: Clarebout, Vlamertinge, Belgium; cv. Saaz: Joh. Barth & Sohn, Nürnberg, Germany) were stored under recommended conditions (cold storage at 0 ◦ C; packaged under vacuum in metallised polyethylene laminates) to avoid deterioration of the brewing principles [6]. Prior to extraction, the hop material was disrupted using a mortar and pestle in order to facilitate subsequent extraction. 2.3. Supercritical fluid extraction (SFE) of hop pellets The vegetative matter (ground pellets) was extracted using a Dionex SFE-703 supercritical fluid extractor (Dionex, Sunnyvale, California, USA). This SFE system is an automated, multi-cell offline extraction instrument (pumping system: 680 atm maximum operating pressure, automated pressure control, automated inlet and vent valves, flow range of 1–18 mL/min liquid CO2 ). The SFE equipment consists of three main parts: a thermostatic sample oven (oven temperature range from 35 to 150 ◦ C) containing up to eight stainless steel extraction cells (cell sizes from 0.5 to 24 mL), a heated flow restrictor (temperature controlled to 250 ◦ C) at the end of each extraction line, and a cooled cryo rack (5 ◦ C ± 3 ◦ C) holding the collection vials (see Fig. 1) [53]. The collection vials are screw-capped glass containers wherein a central inner glass tube is suspended to the closing septum (see Fig. 2). Trapping of

extracted analytes is essentially based on cold solvent trapping, although instant condensation and enrichment of less volatile hop oil constituents invariably occurs at the cold surface of the central inner glass tube [54]. Ethanol (absolute, VWR International, Zaventem, Belgium) was used as trapping solvent to ensure compatibility of the extracts with the beer matrix. Extraction cells (10 mL) were filled with ground hop material (6 g) and placed in the sample oven at a constant temperature (40, 50, or 60 ◦ C, depending on the extraction experiment). The restrictors (flow size: 500 mL) were set at 175 ◦ C to prevent plugging. The SFE extraction was then carried out using carbon dioxide of highest analytical quality (SFE/SFC grade, Air Liquide Benelux, Luik, Belgium) at a specific pressure (80, 85, 90, 95, 100, 105, or 110 atm, depending on the extraction experiment). Extractions were performed until a total volume of 25.0 L of gaseous carbon dioxide was registered by the flow meter of the extractor. Following pressure–temperature combinations were used to extract hop pellets at different CO2 -densities: 90 atm–60 ◦ C (0.20 g/mL); 80 atm–50 ◦ C (0.22 g/mL); 85 atm–50 ◦ C (0.25 g/mL); 100 atm–60 ◦ C (0.29 g/mL); 90 atm–50 ◦ C (0.29 g/mL); 95 atm–50 ◦ C (0.34 g/mL); 100 atm–50 ◦ C (0.39 g/mL); 105 atm–50 ◦ C (0.45 g/mL); 110 atm–50 ◦ C (0.50 g/mL); 90 atm–40 ◦ C (0.50 g/mL); 100 atm–40 ◦ C (0.63 g/mL). 2.4. Preparation of total hop essential oils For isolation of single variety total essential oils, SFE extraction of hop pellets T90 was carried out as described above, applying a pressure of 110 atm and a temperature of 50 ◦ C. After extraction, the collection vial was shaken to dissolve the hop oil constituents that were condensed at the surface of the inner glass tube in the trapping solvent (ethanol). SFE hop oil extract was brought into brown screw-capped glass vials (20 mL) and stored in the freezer at -20 ◦ C until GC-analysis or sensory assessment. 2.5. Preparation of polar hop essence from total hop essential oil

Fig. 2. Construction of the collection vial used in the supercritical fluid extractor [53].

Single variety total hop essential oils were prepared by SFE as described above. Subsequent removal of hydrocarbons (mainly monoterpenes and sesquiterpenes) from total hop oil was achieved via solid phase extraction (SPE). Varian Bond Elut C18 cartridges (500 mg) (Varian, Palo Alto, California, USA) were employed for this purpose. The SPE columns were pre-conditioned with 10 mL HPLCgrade ethanol (LC-grade, Merck, Darmstadt, Germany), followed by 10 mL ethanol/water (1/1; v/v; HPLC-grade ethanol/milli-Q water). Next, total SFE hop oil extract is diluted with milli-Q water (1/1;

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v/v), adsorbed on the column and separated into six fractions (3.0 mL each) by gradually raising the ethanol concentration of the eluent from 50% to 100%. The fraction eluting with 70% ethanol is further indicated as the ‘polar fraction’ or ‘polar essence of total hop essential oil’. Polar essences are stored in the freezer at −20 ◦ C in brown glass vials (20 mL) until GC-analysis or sensory evaluation. 2.6. Continuous steam distillation–continuous extraction (CD-CE) of hop pellets Continuous steam distillation–continuous extraction (CD-CE) of hop pellets was carried out using a micro Likens-Nickerson apparatus (Chrompack, Middelburg, The Netherlands). Hop pellets T90 (2 g) were disrupted using a mortar and pestle prior to extraction and brought into a 100 mL flask containing 60 mL milliQ water. Dichloromethane (2 mL) was brought in a pear-shaped flask (4 mL) to serve as the extraction solvent. Extraction of the volatile hop fraction was carried out by boiling both the hop suspension and dichloromethane under atmospheric pressure during 1 h. The dichloromethane fraction was transferred to a browncoloured glass vial (20 mL) and stored in the freezer (−20 ◦ C) until GC-analysis. 2.7. Gas chromatographic profiling of hop oils The volatile pattern of total hop essential oils and hop oil fractions was studied by capillary gas chromatography on a Thermoquest CE Trace 2000 series, equipped with a flame ionisation detector (GC-FID), a cold-on-column injector and an AS-2000 liquid autosampler (all from Interscience, Louvain-la-Neuve, Belgium). Data acquisition and processing were carried out with the software package ‘Chromcard A/D’, version 1.00. Separation of the volatiles was performed on a fused silica CP-Sil 5 CB column (60 m × 0.25 mm i.d. × 1.0 ␮m film thickness; Chrompack, Middelburg, The Netherlands). CD-CE dichloromethane extract (300 ␮L) or SFE hop oil sample (300 ␮L) was pipetted into a GC vial, spiked with nonadecane (C19, Acros, New Jersey, USA) as internal standard (30 ␮L internal standard solution; 1.50 mg C19/mL ethanol) and subsequently injected cold-on-column into the gas chromatograph. Helium was used as carrier gas (flow rate: 1 mL/min). The column oven temperature was programmed from 40 ◦ C to 260 ◦ C at 10 ◦ C increase per minute with a period of 16 min at the final temperature. The FID detector base was set at 290 ◦ C. A mixture of reference compounds (␤-caryophyllene, caryophyllene oxide, ␣-copaene, ␣-humulene, linalool, and 2undecanone) was injected (injection volume: 1 ␮L; standard solution containing ± 0.5 ppm of each compound in ethanol) for identification of marker constituents in extracts and essences. Other hop oil constituents (methyl 4-decenoate, humulene epoxide I, humulene epoxide II, humulenol II, and ␤-selinene), were tentatively identified on the basis of retention indices found in literature databases [55,56]. 2.8. Brewing of reference lager beer A reference pilot lager beer was prepared at our pilot brewery (2 hL scale). To prevent introduction of hoppy aroma in view of reliable sensory evaluation of the flavour effects of the different hop oil preparations, the reference beer was exclusively bittered by the addition of pre-isomerised hop extract (Botanix, Kent, UK) at the end of wort boiling. Brewing was performed with 34 kg pilsner malt, 6 kg maize flakes and 1.4 hL reverse osmosis brewing water (with the addition of CaCl2 (40 mg/L)). The brewing scheme was as follows: mashingin at 36 ◦ C (15 min), followed by successive temperature increases (rise in temperature: 1 ◦ C/min) to 49 ◦ C (30 min), 63 ◦ C (20 min),

72 ◦ C (30 min), and 78 ◦ C (120 min, including wort filtration with lauter tun and sparging until 11.5 P at the onset of boiling). Wort boiling was done at atmospheric pressure for 75 min (8% of evaporation), followed by wort clarification in the whirlpool. Bittering was performed by adding 3.85 g iso-␣-acids/hL at the end of wort boiling (presumed utilisation: 65%) to achieve 25 ppm iso-␣-acids in the final beer. Original gravity: 12.0 P; pitching rate: 107 yeast cells/mL; fermentation temperature and duration: 12 ◦ C, 8 days; lagering: 10 days at 0 ◦ C; beer filtration: kieselguhr/cellulose sheets (1 ␮m); packaging: automatic filling with pre-evacuation and carbon dioxide flushing (6-head America monobloc filling equipment, Cimec, Italy) in brown glass bottles (25 cL) closed with conventional crown corks. Characteristics of the reference beer were determined via standard analyses according to EBC methods: alcohol: 5.15% (v/v); apparent extract: 2.49 g/100 g; original extract: 12.0 g/100 g; pH: 4.31; dissolved oxygen: 54 ␮g/L; carbon dioxide: 5.6 g/L; soluble proteins: 314 mg/L; total polyphenols: 93.2 mg/L; FAN: 42.5 mg/L; iso-␣-acids: 24.4 ppm. 2.9. Sensory analyses For sensory evaluation, the hop oil preparations were added to a non-aromatised pilot lager. Levels of addition were calculated on the basis of GC-FID analysis of the respective hop oil. For total hop essential oils, addition levels are based on the total amount of all volatiles present in the GC-FID pattern (level of addition: 0.05, 0.10, 0.25, 0.50 or 1.00 ppm, depending on the experiment). For polar hop essences, levels of addition are based on the amount of the sesquiterpenoid (‘spicy’) fraction present in the GC-FID trace of polar essence (levels of addition: 2.5, 5, 10, 20 or 40 ppb of ‘spicy’ fraction, depending on the experiment). Sensory analyses were performed by 12 members of a trained taste panel. Triangular tests were carried out to evaluate whether aromatisation of the reference beer with the respective hop oil preparations significantly affects the flavour profile, to assess potential varietal typicality of the hop oil preparations, and to estimate the flavour threshold value of total hop essential oil or polar hop essence in beer. Varietal typicality of the hop oil preparations was assessed via 6 triangular tests performed in 6 separate sessions. In each session, 3 aromatised pilot lager beers (2 beers aromatised with the same variety, 1 beer aromatised with another variety) were presented to the taste panel and it was asked to select the different sample. The following varietal comparisons were made: Saaz vs. East Kent Golding, Saaz vs. Hersbrucker Spät, East Kent Golding vs. Hersbrucker Spät. Total hop oils were added at 0.50 ppm; polar hop essences were added at 20 ppb ‘spicy’ fraction. In addition, panellists were also asked to give their preference for a particular sample. The preference was taken into account when the triangular test showed significant difference between the samples. Flavour threshold values of varietal total hop essential oils in beer were determined by the ‘ascending method of limits’ [57]. This method has been used for example by Peacock et al. [58] to determine flavour thresholds of floral compounds in beer. Five sets of three glasses were presented to each assessor. Of each triangular set, only one beer was aromatised with total hop essential oil at a level of 50, 100, 250, 500, and 1000 ppb, respectively. The instruction was to select the sample with addition of total hop essential oil from each triangular set. The sensory test started with the lowest concentration set. Calculation of the ‘best estimate threshold’ (BET) for each individual panellist is based on the geometric mean of the lowest concentration that was detected by the panellist and the next concentration in the test series. A group threshold is then calculated as the geometric mean of the individual BETs. The above described procedure was repeated for estimation of the flavour

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Fig. 3. GC-FID profile of total hop oil cv. East Kent Golding obtained by SFE (110 atm, 50 ◦ C) (top) and continuous steam distillation–continuous extraction (CD-CE) (bottom). (s: solvent impurity; a: ␤-myrcene; b: linalool; c: 2-undecanone; d: methyl 4-decenoate; e: ␣-copaene; f: ␤-caryophyllene; g: ␣-humulene; h: ␤-selinene; i: caryophyllene oxide; j: humulene epoxide I; k: humulene epoxide II; l: unidentified sesquiterpenoid; m: humulenol II; IS: internal standard, nonadecane).

threshold value for varietal polar hop essences. Addition level of polar essence was as follows: 2.5, 5, 10, 20, and 40 ppb of ‘spicy’ fraction. For descriptive sensory analysis of beers aromatised with single variety total hop essential oils, three separate taste sessions were performed. In a first session, the non-aromatised pilot lager and pilot lager aromatised with total hop essential oil cv. Saaz (level of addition: 500 ppb total volatiles) were presented to the members of the taste panel (12 panellists). Panellists were asked to describe sensory impressions related to taste, aroma, and mouthfeel, and to score the intensity of the descriptors on a scale ranging from 0 (not perceptible) to 8 (very high intensity). In a second and third session, the descriptive sensory test was repeated for beers aromatised with total hop essential oil from cv. East Kent Golding and cv. Hersbrucker Spät. The above procedure for descriptive sensory analysis was also applied to pilot lagers aromatised with polar hop essences cv. Saaz, cv. East Kent Golding, and cv. Hersbrucker Spät (level of addition: 20 ppb ‘spicy’ fraction). 2.10. Multivariate data analysis by principal component analysis Principal component analysis (PCA) was carried out to enhance data analysis and for interpretation of the results. In this study, PCA was used for discrimination of single variety SFE/SPE total hop

essential oils or polar hop essences on the basis of their volatile analytical GC-FID pattern. PCA was performed by using the multivariate data analysis software package The Unscrambler® v9.2 (CAMO, Oslo, Norway). 3. Results and discussion 3.1. Single variety total hop essential oils: extraction via SFE and analytical evaluation In order to determine the best experimental conditions required for isolation of total hop essential oils from hop pellets, SFE extractions were carried out using supercritical carbon dioxide at different pressure-temperature combinations, i.e. different carbon dioxide densities. Firstly, it is explained how the hop oil extracts will be evaluated on the basis of GC-FID analysis. A typical GC-FID pattern of total hop oil obtained by SFE (110 atm, 50 ◦ C) is shown in Fig. 3 (upper chromatogram). The analytical profile shows the characteristic composition of a total hop oil which is dominated by the monoterpene ␤-myrcene (1) and the sesquiterpenes ␤-caryophyllene (2) and ␣-humulene (3) (for structural formula of components, see Fig. 4). For detailed evaluation of the experimental conditions for SFE of total hop oils from hop pellets, the chromatogram in Fig. 3 is further subdivided into three different parts, each part

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(1)

(2)

(3)

(4)

O

O

O

(5)

(6)

(7)

(8)

O

O

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O

(9)

(10)

(11)

(12)

Fig. 4. Structures of representative markers of the floral, sesquiterpene hydrocarbon, and spicy fraction of hop essential oil.

data [48,63], the lower carbon dioxide densities are insufficient to isolate total hop oils. For instance, whereas, at a carbon dioxide density of 0.34 g/mL, the most volatile fraction, i.e. the floral part of hop oil, is already fully extracted, only about 80% and 25% of the sesquiterpene hydrocarbons and the oxygenated sesquiterpenoids (spicy fraction), respectively, are obtained. At the lower carbon dioxide densities of 0.22 g/mL and 0.25 g/mL, almost no oxygenated sesquiterpenoids are found in the extracts. In summary, our data in Fig. 5 and in Table 1 demonstrate that SFE of total hop essential oil requires application of a sufficiently high carbon dioxide density (0.50 g/mL). Due to the increasing solubility power of carbon dioxide at higher solvent densities, some contamination of SFE hop oil extracts with non-volatile components, in particular hop acids, may occur. Therefore, in order to limit co-extraction of hop acids, the pressuretemperature combination of 110 atm and 50 ◦ C was taken as the standard for isolation of total hop essential oils. In this way, under the applied experimental conditions for SFE, less than 10% of hop acids present in the sample were extracted (data not shown), whereas hop oils represent the full spectrum of highly volatile and less volatile constituents.

Sum of marker compounds (μg/g hops)

representing a group of hop oil constituents. Next, representative marker compounds of each group were selected in order to determine the efficiency of the extraction experiments in relation to the three different groups of hop oil components. The first section of the chromatogram is indicated as the ‘floral’ part because it contains monoterpenoids and oxygenated compounds associated with ‘floral’ hop aroma impressions [20,58–60]. Selected marker components for this group are the monoterpene alcohol linalool (4), 2-undecanone (5), and methyl 4-decenoate (6). The second part is indicated as the sesquiterpene hydrocarbon fraction (SHC) since it consists of sesquiterpene hydrocarbons (selected markers: ␣-copaene (7), ␤-caryophyllene, ␣-humulene, and ␤-selinene (8)). The third section of the chromatogram is indicated as the ‘spicy’ fraction because it contains mainly sesquiterpenoids that have been linked with the ‘spicy’, ‘noble’ hop character [17,19,21,22,58,61,62]. Selected marker components for the ‘spicy’ region are caryophyllene oxide (9), humulene epoxide I (10), humulene epoxide II (11), humulenol II (12) and an unidentified sesquiterpenoid. The results of the different SFE extractions carried out at different pressure-temperature combinations are presented as GC-FID semi-quantitative data obtained on the above mentioned marker components, representing the floral, sesquiterpene hydrocarbon, and spicy part of hop essential oil. Fig. 5 shows the total level, i.e. the sum of marker volatiles extracted from hop pellets T90 cv. Saaz, as a function of the applied carbon dioxide density. On analysis of the complete series of these extractions (0.20 g/mL to 0.63 g/mL) it can be concluded that, for the production of a total hop essential oil, a carbon dioxide density of 0.50 g/mL is the minimum requirement. The total level of selected marker compounds (expressed as ␮g/g hop pellets) extracted at this density is indeed significantly higher than that obtained at lower densities. Interestingly, at the lower carbon dioxide densities (0.20–0.25 g/mL), only a small fraction, i.e. about 20%, of total hop oil is extracted. This finding is in clear contrast with several data found in literature, reporting that relatively low carbon dioxide densities of 0.20 g/mL and 0.25 g/mL are already sufficient for selective isolation of total hop oils from dried hop cones or hop pellets [48,63]. On the other hand, Zekovic´ et al. [64] applied a carbon dioxide density of 0.79 g/mL (extraction at 150 atm and 40 ◦ C) for isolation of total hop essential oil as the first step in a two-stage SFE procedure comprising successive extraction of the aroma fraction and the bittering principles from dried hop cones. The contents of selected marker components in hop oils prepared at 50 ◦ C and extraction pressures ranging from 80 atm to 110 atm are shown in Table 1. Obviously, in contrast to literature

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10

11

SFE extracons at different pressure-temperature combinaons Fig. 5. Total mass of marker volatiles extracted from 1 g of hop pellets T90 cv. Saaz at different carbon dioxide densities (1 = 0.20 g/mL, 90 atm, 60 ◦ C; 2 = 0.22 g/mL, 80 atm, 50 ◦ C; 3 = 0.25 g/mL, 85 atm, 50 ◦ C; 4 = 0.29 g/mL, 100 atm, 60 ◦ C; 5 = 0.29 g/mL, 90 atm, 50 ◦ C; 6 = 0.34 g/mL, 95 atm, 50 ◦ C; 7 = 0.39 g/mL, 100 atm, 50 ◦ C; 8 = 0.45 g/mL, 105 atm, 50 ◦ C; 9 = 0.50 g/mL, 110 atm, 50 ◦ C; 10 = 0.50 g/mL, 90 atm, 40 ◦ C; 11 = 0.63 g/mL, 100 atm, 40 ◦ C). Error bars represent mean ± standard deviation (3 extractions at each pressure–temperature combination).

F. Van Opstaele et al. / J. of Supercritical Fluids 69 (2012) 45–56

51

Table 1 Yield of hop oil marker components (expressed as ␮g/g hops, mean of 3 extractions) present in the SFE extracts prepared at 50 ◦ C in combination with different pressures from hop pellets. T90 cv. Saaz. (crop year 2005, Clarebout, Belgium). (n.d.: not detected; S.D.: standard deviation; CV(%): coefficient of variation). Marker components

Extraction pressure (atm)

Floral fraction Linalool 2-Undecanone Methyl 4-decenoate Sesquiterpene hydrocarbon fraction ␣-Copaene ␤-Caryophyllene ␣-Humulene ␤-Selinene Spicy fraction Caryophyllene oxide Humulene epoxide I Humulene epoxide II Humulenol II Unknown sesquiterpenoid Sum marker compounds S.D. CV (%)

80 85 Carbon dioxide density (g/mL)

90

95

100

105

110

0.22

0.25

0.29

0.34

0.39

0.45

0.50

4.5 1.7 2.9

7.2 4.3 6.0

3.0 99.2 175.3 9.7 0.5 0.4 2.3 0.2 n.d. 299.2 18.0 6.0

15.0 19.3 14.2

19.1 28.0 20.0

18.1 29.1 18.2

23.2 30.5 19.3

17.8 32.4 18.9

3.3 128.0 215.7 12.2

6.4 324.2 525.2 31.0

7.6 475.2 739.1 42.8

8.3 430.9 792.6 61.6

11.0 465.2 870.6 63.7

12.2 505.3 961.0 77.9

0.6 0.4 3.1 n.d. n.d. 380.8 22.1 5.8

2.9 2.4 18.0 1.9 3.9 964.4 40.4 4.2

3.6 2.8 21.8 1.3 6.0 1367 95.7 7.0

9.1 8.1 52.5 6.1 27.1 1462 102.0 7.0

11.4 10.0 62.0 8.0 29.9 1605 93.4 5.8

12.8 12.1 73.6 8.9 40.5 1773 92.1 5.2

Clearly, besides the applied carbon dioxide density, also the extraction time is very important for SFE recovery of total hop oil. Fig. 6 displays the extraction behaviour of the selected hop oil markers as a function of SFE extraction time (carbon dioxide density: 0.50 g/mL). It appears that most volatile marker components of the floral region (linalool, 2-undecanone, methyl 4-decenoate) are already effectively extracted at the beginning of the extraction process, i.e. after 20–30 min these compounds are totally extracted. However, extraction has to be prolonged considerably for sufficient isolation of both the sesquiterpene hydrocarbons (e.g. ␣-humulene) and the oxygenated sesquiterpenoids (e.g. humulene epoxide II) since their relative amount becomes approx. 0% and 10%, respectively, after 90 min of extraction time. These findings on differential SFE extraction behaviour of hop oil components, as shown in Fig. 6, are furthermore in line with literature data as reported by Pourmortazavi and Hajimirsadeghi [54]. In their review on supercritical fluid extraction of plant essential oils, these authors explain that extraction of monoterpenes takes place at the beginning of the extraction process, whereas sesquiterpenes and oxygenated components need longer extraction times as a result of their higher molecular weight and polarity. Further SFE experiments at a carbon dioxide density of 0.50 g/mL showed that hop oxygenated sesquiterpenoids were fully extracted

after 125 min (data not shown). Thus, when aiming at SFE preparation of total hop oil, 125 min is the minimal extraction time under the applied experimental conditions. The use of a 500 mL flow size restrictor in the extraction equipment corresponds in practice with a carbon dioxide flow of 200 mL/min on average, which also implies that the extraction can be regarded as completely finished when 25.0 L of gaseous carbon dioxide has been collected. The extent of reproducibility of SFE preparation of total hop essential oil under the optimised experimental conditions (110 atm, 50 ◦ C, extraction time: 125 min) is shown in Table 2. The relatively low coefficients of variation obtained on the individual marker components (mostly less than 8%) and on the sum of marker volatiles (3.9%) demonstrate satisfying reproducibility of the applied SFE procedure. Furthermore, comparison of the SFE methodology with classic Likens–Nickerson continuous distillation–continuous extraction (CD-CE) shows no qualitative differences between the volatile GC-FID patterns of hop essential oils prepared via SFE and CD-CE (see Fig. 3). Reproducibility of the optimised SFE procedure for the preparation of single variety total hop essential oils was further evaluated by repeated extractions (5 times) on pellets T90 from different hop varieties, i.e. cv. Saaz, cv. East Kent Golding (EKG), and cv. Hersbrucker Spät. These results are shown in Table 3. The total level of

100 linalool

90 2-undecanone

Relave amount (%)

80 methyl 4-decenoate

70 alpha-copaene

60 beta-caryophyllene

50 alpha-humulene

40 beta-selinene

30 caryophyllene oxide

20

humulene epoxide I

10

humulene epoxide II

0 10

20

30

45

60

75

90

humulenol II

Extracon me (min) Fig. 6. Relative amount of selected hop oil marker components as a function of SFE extraction time (SFE extraction of hop pellets T90 cv. Saaz at 110 atm and 50 ◦ C).

52

F. Van Opstaele et al. / J. of Supercritical Fluids 69 (2012) 45–56

Table 2 Reproducibility of SFE preparation of total hop essential oil from pellets T90 cv. Saaz (crop year 2005; Joh. Barth & Sohn, Germany) (five extraction experiments were carried out at 110 atm and 50 ◦ C; levels of hop oil marker components are expressed as ␮g/g hops; S.D.: standard deviation; CV: coefficient of variation). Marker components

Extraction experiment 1

Floral fraction Linalool 2-Undecanone Methyl 4-decenoate Sesquiterpene hydrocarbon fraction ␣-Copaene ␤-Caryophyllene ␣-Humulene ␤-Selinene Spicy fraction Caryophyllene oxide Humulene epoxide I Humulene epoxide II Humulenol II Unknown sesquiterpenoid Sum marker volatiles

Mean

2

3

4

S.D.

CV (%)

5

11.7 28.3 15.2

12.7 29.5 18.1

13.7 30.1 19.4

12.4 28.8 16.8

13.2 29.7 17.5

12.7 29.3 17.4

0.7 0.7 1.6

5.5 2.4 9.2

11.4 444.0 862.5 79.4

10.5 507.2 987.0 83.7

11.7 481.5 933.2 80.1

10.8 472.5 897.6 81.5

11.4 484.3 921.1 82.1

11.2 477.9 920.3 81.4

0.5 22.9 46.0 1.7

4.5 4.8 5.0 2.1

7.8 10.6 64.3 7.6 55.8 1599

8.3 10.2 56.7 7.2 45.1 1776

8.6 9.0 51.8 6.6 36.6 1682

8.2 9.4 56.7 7.1 40.7 1643

7.9 10.3 60.5 7.2 42.3 1688

8.2 9.9 58.0 7.1 44.1 1677

0.3 0.7 4.7 0.4 7.2 65.8

3.6 7.1 8.1 5.6 16.3 3.9

extracted volatiles, i.e. the level of total hop oil, was estimated on the basis of all peaks in the corresponding GC-FID-profile, whereas for determination of the sum of all volatiles present in the floral, sesquiterpene hydrocarbon, and spicy fraction, GC chromatograms were subdivided as was done for Fig. 3. Satisfying reproducibility is observed for the SFE extractions since coefficients of variation for the level of total hop oils range from 7% to 11%. Furthermore, clear differences are noticed among the different hop varieties. The yield of extracted volatiles is significantly higher for cv. EKG and cv. Hersbrucker Spät than for cv. Saaz. A comparable total level of volatiles is found for cv. EKG and cv. Hersbrucker Spät (7269 and 7510 ␮g/g hops, respectively). However, clear differences are observed in the relative composition of the extracts. Whereas the floral fraction for cv. EKG amounts to more than 50% of total hop oil, the relative proportion of spicy components (oxygenated sesquiterpenoids) is much higher for cv. Hersbrucker Spät than for cv. EKG. Similar to cv. EKG, hop oil of cv. Saaz is relatively rich in floral constituents (approx. 60%), but, at the same time, it contains a considerable proportion of oxygenated sesquiterpenoids (12.9%). With regard to the ‘spicy’ part of hop oil, the level of oxygenated sesquiterpenoids is the highest for cv. Hersbrucker Spät. In accordance with this, literature data report on the characteristic sesquiterpenoid fraction of the Hersbrucker Spät variety [65] and the pronounced spicy character of Hersbrucker hopped beers [17,22,49,50]. Analytical differences between the single variety total hop oil extracts derived from the varieties Saaz, East Kent Golding and Hersbrucker Spät, are further demonstrated in Fig. 7 via principal component analysis on a data matrix, composed of the prepared varietal hop oils (objects) and approx. 30 different volatiles (variables), determined by GC-FID. The bi-plot in Fig. 7, shows that 97% of the variance is explained by the first two principal components and, furthermore, the pure varietal hop oils are clearly differentiated on the basis of their volatile pattern determined by GC-FID. Total hop oil cv. Hersbrucker Spät is mainly characterised by the oxygenated sesquiterpenoids (caryophyllene oxide, humulene epoxide I, and humulene epoxide II). On the other hand, the major sesquiterpene

hydrocarbons ␤-caryophyllene and ␣-humulene clearly correlate with the variety EKG, whereas methyl 4-decenoate and ␣-copaene are more typical for the volatile pattern of hop oil cv. Saaz. 3.2. Sensory assessment of single variety total hop essential oils Upon smelling of the total hop oils as such, intense odours, described as ‘hoppy’, ‘pellet-like’, and ‘floral’ were noted for all hop oils. For evaluation of flavouring effects in the beer matrix, a reference pilot lager beer, exclusively bittered with pre-isomerised hop extract, was aromatised with precise amounts of the varietal total hop oil preparations. Levels of addition were calculated on the basis of the total amount of volatiles in the respective hop oils, as determined by GC-FID. First, beer samples were aromatised by adding a particular volume of total hop oil cv. Saaz containing 50 ␮g, 100 ␮g, 250 ␮g, 500 ␮g, or 1000 ␮g of total volatiles to 1 l of the finished reference beer, i.e. levels of addition ranged from approx. 0.05 ppm to 1.00 ppm. Sensory assessment via the ascending method of limits [57] using triangular tests (see Table 4) shows that the hop oil cv. Saaz had a clear impact on the flavour of the reference beer when added at 0.50 ppm since all panellists were able to differentiate the beers. Table 4 further shows that the aromatised beer was preferred by most panellists when the hop oil was added at 0.50 ppm to the reference beer. A higher addition level (1.00 ppm) was found to have a too high impact on the beer flavour. The flavour threshold value for this particular total hop oil in the experimental pilsner beer amounts to 0.26 ppm as could be determined from the individual ‘best estimate thresholds’. Further descriptive sensory analysis demonstrated that the flavour profile of the reference beer changed significantly by adding 0.50 ppm of total hop oil cv. Saaz, cv. EKG, or cv. Hersbrucker Spät (see Fig. 8). Next to a clear hoppy and citrusy aroma, a higher bitterness intensity, and an increase in mouthfeel and fullness are the most prominent sensory changes denoted for all aromatised beers. An interesting observation is also that the unpleasant malty flavour of the non-aromatised reference beer is fully masked by addition of

Table 3 Yield of extracted volatiles determined by GC-FID and relative composition (%) of total varietal hop essential oils obtained by SFE (110 atm; 50 ◦ C) of pellets T90 from hop varieties Saaz, East Kent Golding, and Hersbrucker Spät. (CV: coefficient of variation; 5 extractions). Hop variety

Saaz East Kent Golding Hersbrucker Spät

Total

Floral fraction

SHC fraction

Spicy fraction

␮g/g hops

CV (%)

␮g/g hops

%

␮g/g hops

%

␮g/g hops

%

4822 7269 7510

7.0 11.1 9.2

2874 3831 3124

59.6 52.7 41.6

1326 3155 3327

27.5 43.4 44.3

622 283 1059

12.9 3.9 14.1

F. Van Opstaele et al. / J. of Supercritical Fluids 69 (2012) 45–56

53

Fig. 7. Bi-plot of principal component analysis on single variety total hop essential oils obtained by SFE of pellets T90 cv. Saaz, cv. East Kent Golding (EKG), and cv. Hersbrucker Spät (Hersb Sp), respectively. Varietal SFE hop oils are represented as scores and volatiles determined by GC-FID as loadings (3 extracts were prepared per variety; numbering of variables (italics) refers to unidentified components).

(A)

(B)

citrus

citrus 7

7

6

6

fullness

fullness

fruity

5

4

3

3

2

2

fruity

1

1

mouthfeel

5

4

floral

0

bierness intensity

mouthfeel

bierness intensity

malty

floral

0

malty

Blank

hoppy

(C)

Blank + total hop oil cv. Saaz

hoppy

Blank Blank + total hop oil cv. EKG

citrus 7 6

fullness

5

fruity

4 3 2 1

mouthfeel

floral

0

bierness intensity

malty

hoppy

Blank Blank + total hop oil cv. Hersb. Sp.

Fig. 8. Spider diagrams representing aromatisation of a reference pilot lager (blank) with SFE total hop essential oil from cv. Saaz (A), cv. EKG (B), and cv. Hersbrucker Spät [Hersb. Sp.; (C)], respectively (addition rate: 0.50 ppm of total hop essential oil).

54

F. Van Opstaele et al. / J. of Supercritical Fluids 69 (2012) 45–56

Table 4 Sensory impact of total hop essential oils prepared by SFE on the flavour of a pilot lager (Blank: non-aromatised reference lager; ˛: significance level according to Meilgaard et al. [57]). Triangular test Evaluation of the level of addition Blank vs. aromatised cv. Saaz (0.05 ppm) Blank vs. aromatised cv. Saaz (0.10 ppm) Blank vs. aromatised cv. Saaz (0.25 ppm) Blank vs. aromatised cv. Saaz (0.50 ppm) Blank vs. aromatised cv. Saaz (1.00 ppm) Evaluation of varietal aspect (aromatisation: 0.50 ppm) Saaz vs. East Kent Golding Saaz vs. Hersbrucker Spät East Kent Golding vs. Hersbrucker Spät a

Number of correct answers (12 panellists) and ˛-level

Preference

2 (˛ > 0.4) 5 (˛ = 0.4) 7 (˛ = 0.10) 12 (˛ = 0.00) 12 (˛ = 0.00)

– – – Blank: 4a ; Aromatised: 8 Blank: 8; Aromatised: 4

10 (˛ = 0.001) 10 (˛ = 0.001) 11 (˛ < 0.001)

Saaz: 7; East Kent Golding 3 Saaz: 3; Hersbrucker: 7 East Kent Golding: 2; Hersbrucker: 9

Number of panellists preferring the sample.

the hop oils. Fig. 8 further shows the clear impact of the hop variety on the beer flavour profile. For example, fruity aroma is lower when the pilot beer is aromatised with total hop oil cv. Saaz or cv. EKG compared to aromatisation with hop oil cv. Hersbrucker Spät, whereas aromatising the reference beer with total hop oil cv. EKG has the most pronounced effect on citrusy aroma. Convincing evidence for sensory differences when adding different varietal total hop oils to beer was further obtained through triangular tests (see Table 4, evaluation of varietal aspect). Beer samples aromatised with 0.50 ppm of varietal total hop oil cv. Saaz, cv. East Kent Golding, and cv. Hersbrucker Spät, were all significantly discriminated by our taste panel and, obviously, beers aromatised with hop oil cv. Hersbrucker Spät were preferred by the panellists. Although, in respect of overall beer flavour quality, total hop oil additions were evaluated positively, the members of the taste panel also agreed that the added hop aromas imparted some negative influence on the quality of beer bitterness (somewhat harsher) and mouthfeel (more pungent aftertaste). 3.3. Single variety polar hop essences: preparation and analytical/sensory evaluation Total hop essential oils were extracted from pellets T90 from different varieties (cv. Saaz, cv. East Kent Golding, cv. Hersbrucker Spät) by one-step SFE as described before. In order to prepare varietal, fractionated hop oils comprising the water-soluble, oxygenated fraction of total essential oil, i.e. single variety polar hop essences, a chromatographic method based on SPE was developed. Total hop oil was brought on top of a pre-conditioned C18 SPE cartridge which was then eluted with ethanol/water mixtures of increasing ethanol concentration. The hop oil fraction eluting with 70/30 (v/v) ethanol/H2 O showed a pleasant, hoppy fragrance as such and, upon addition to beer, pronounced hoppy character was noticed. Therefore, the volatile composition of this fraction was further investigated via GC-FID. The typical composition of varietal polar hop essences, as determined by GC-FID, is shown in Table 5. Clearly, hop aromas highly enriched in both the floral and spicy fraction of total hop oil are obtained because the non-polar sesquiterpene hydrocarbons are

virtually removed by the SPE fractionation process. On account of this characteristic composition (approx. 80% ‘floral’ fraction and 20% ‘spicy’ fraction, see Table 5), these hop oil preparations are therefore indicated as the polar fraction of total hop oil or ‘polar hop essence’. Preliminary tasting sessions were performed by adding varying amounts of polar hop essence cv. Saaz to a nonaromatised reference pilot pilsner. Panellists agreed that additions of hop essence cv. Saaz corresponding to 20 ␮g of ‘spicy’ fraction to 1 litre of finished beer resulted in clear sensory effects with regard to the aroma and mouthfeel. Furthermore, at higher levels, flavour impressions introduced by adding polar hop essence cv. Saaz to the pilot pilsner, were considered as overwhelming. As subsequently determined via the ascending method of limits using triangular tests (see Section 2) the flavour threshold value for the polar hop essence cv. Saaz in the pilot lager amounted to 14.1 ppb (corresponding to ‘spicy’ fraction). In addition, flavour threshold values of the polar hop essences cv. EKG and cv. Hersbrucker Spät were determined at 15.5 ppb and 9.8 ppb, respectively. Further descriptive sensory analysis demonstrated significant changes in the flavour profile of the reference beer when it was aromatised with an adequate amount (level of addition corresponding to 20 ppb ‘spicy’ fraction) of varietal polar hop essence (see Fig. 9). Next to a pronounced, pleasant hoppy aroma, a higher bitterness and increased mouthfeel and fullness are the most prominent sensory changes perceived in all aromatised beers. Interestingly, the malty flavour of the non-aromatised reference beer is fully masked by the addition of polar hop essence. These findings are furthermore analogous to the sensory results obtained from aromatising the reference pilot lager with single variety total hop essential oil. Evidence for sensory differences when adding different varietal polar hop essences to beer was obtained through triangular tests (for experimental details, see Section 2). Beer samples aromatised with single variety polar hop essences prepared from pellets T90 cv. Saaz, cv. East Kent Golding, and cv. Hersbrucker Spät, were all significantly discriminated by the taste panel (␣ ≤ 0.001). Furthermore, in contrast to total hop oils, the polar hop essences did not impart any negative effect on the quality of beer bitterness and mouthfeel.

Table 5 Yield of volatiles determined by GC-FID and relative composition of single variety polar hop essences obtained by SPE fractionation of total hop essential oils (SFE/SPE was performed 3 times on each hop variety; levels of volatiles are expressed as mean values ± standard deviation; relative composition expressed as a percentage of total amount of volatiles in polar hop essence). Hop variety

Saaz East Kent Golding Hersbrucker Spät

Floral fraction

SHC fraction

Spicy fraction

␮g/g hops

%

␮g/g hops

%

␮g/g hops

%

198.1 ± 10.1 244.6 ± 15.9 704.2 ± 32.7

77.7 75.4 80.3

5.4 ± 0.4 14.2 ± 0.6 14.5 ± 0.7

2.1 4.4 1.6

51.3 ± 1.8 65.7 ± 3.2 158.2 ± 9.9

20.1 20.2 18.0

F. Van Opstaele et al. / J. of Supercritical Fluids 69 (2012) 45–56

(A)

(B)

citrus

citrus

7

7 6

6

fullness

5

fullness

fruity

5

4

4

3

3

2

2

floral

0

bierness intensity

mouthfeel

floral

0

bierness intensity

malty

malty Blank

Blank

hoppy

hoppy

(C)

fruity

1

1

mouthfeel

55

Blank + polar essence cv. EKG

Blank + polar essence cv. Saaz

citrus 7 6

fullness

5

fruity

4 3 2 1

mouthfeel

floral

0

bierness intensity

malty Blank

hoppy

Blank + polar essence cv. Hersb. Sp.

Fig. 9. Spider diagrams representing aromatisation of a reference pilot lager (Blank) with SFE/SPE polar hop essences from cv. Saaz (A), cv. EKG (B), and cv. Hersbrucker Spät (Hersb. Sp.; (C)), respectively (addition rate corresponding to 20 ppb ‘spicy’ fraction of polar hop essence).

4. Conclusions

References

Single variety total hop essential oils were extracted directly from pellets T90 by supercritical fluid extraction (SFE) using carbon dioxide. A carbon dioxide density of 0.50 g/mL, i.e. a pressuretemperature combination of 110 atm and 50 ◦ C, proved to be the best compromise for selective isolation of hop oils comprising the full spectrum of volatiles, including oxygenated sesquiterpenoids. Single variety polar hop essence was obtained by further SPE fractionation of total hop essential oil on C18 silica. The hop oil fraction eluting with 70/30 (v/v) ethanol/water showed pleasant hoppy character when added to beer and was therefore selected for further investigation. From an analytical point of view, non-polar sesquiterpene hydrocarbons are largely removed during the SPE fractionation step. Consequently, a hop oil fraction highly enriched in ‘floral’ and ‘spicy’ compounds is obtained. Sensory evaluations demonstrated that the addition of total SFE hop oil and polar hop oil essence significantly changes the flavour profile of a reference pilot lager. A pleasant, intense hoppy aroma, and a rise in bitterness intensity and mouthfeel are the most striking sensory contributions when added to beer. Single variety hop oil additions were significantly discriminated by the taste panel, which may reflect analytical differences in the composition of the hop oils.

[1] M. Moir, Hops—a millennium review, J. American Society of Brewing Chemists 58 (4) (2000) 131–146. [2] M. Verzele, Centenary review—100 years of hop chemistry and its relevance to brewing, J. Institute of Brewing 92 (1986) 32–48. [3] S. Langstaff, M.J. Lewis, Mouthfeel of beer—a review, J. Institute of Brewing 99 (1993) 31–37. [4] A. Forster, R. Schmidt, The characterization and classification of hop varieties, in: EBC Monograph 22, Fachverlag Hans Carl, Nürnberg, 1994, pp. 251–269. [5] A. Forster, B. Beck, R. Schmidt, Untersuchungen zu Hopfenpolyphenolen, in: Proceedings of the 25th Congress of the European Brewery Convention, Oxford University Press, Oxford, 1995, pp. 143–150. [6] J.L. Benitez, A. Forster, D. De Keukeleire, M. Moir, F.R. Sharpe, L.C. Verhagen, K.T. Westwood, European brewery convention manual of good practice: hops and hop products, Fachverlag Hans Carl, Nürnberg, 1997. [7] K. Goiris, E. Syryn, B. Jaskula, F. Van Opstaele, G. De Rouck, G. Aerts, L. De Cooman, Hop polyphenols: potential for beer flavour and flavour stability, in: Proceedings of the 30th Congress of the European Brewery Convention, Fachverlag Hans Carl, Nürnberg, Contribution 87, 2005, pp. 1–13. [8] I.R. McLaughlin, C. Lederer, T.E. Shellhammer, Bitterness-modifying properties of hop polyphenols extracted from spent hop material, J. American Society of Brewing Chemists 66 (3) (2008) 174–183. [9] M. Verzele, D. De Keukeleire, Chemistry and Analysis of Hop and Beer Bitter Acids, Elsevier Science, Amsterdam, 1991. [10] B. Jaskula, P. Kafarski, G. Aerts, L. De Cooman, A kinetic study on the isomerization of hop ␣-acids, J. Agricultural and Food Chemistry 56 (2008) 6408–6415. [11] A. Forster, Trends in the production of non-isomerised hop products, in: EBC Monograph 22 Fachverlag Hans Carl, Nürnberg, 1994, pp. 72–87. [12] G.W. Smith, Isomerised hop products, in: EBC Monograph 22, Fachverlag Hans Carl, Nürnberg, 1994, pp. 92–103. [13] M. Biendl, Pre-isomerised hop products–potential and practical use: an up-todate overview, Brauwelt International IV (2002) 20–25. [14] L. De Cooman, G. Aerts, F. Van Opstaele, K. Goiris, E. Syryn, G. De Rouck, M. De Ridder, D. De Keukeleire, New trends in advanced hopping—part 1: application of pre-isomerised hop extracts, Cerevisia 29 (1) (2004) 36–46. [15] L. De Cooman, G. Aerts, H. Overmeire, D. De Keukeleire, Alterations of the profiles of iso-␣-acids during beer ageing, marked instability of trans-iso-␣-acids and implications for beer bitterness consistency in relation to tetrahydroiso␣-acids, J. Institute of Brewing 106 (2000) 169–178.

Acknowledgements We would like to thank all members of the taste panel of our institute. HVG – Hopfenverwertungsgenossenschaft e.G. (Wolnzach, Germany) is thanked for financial support of this study.

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