Bio Transformation of Geraniol

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The Contribution of Geraniol Metabolism to the Citrus Flavour of Beer: Synergy of Geraniol and β-Citronellol Under

Coexistence with Excess Linalool

Kiyoshi Takoi1,2,*, Yutaka Itoga3, Koichiro Koie3, Takayuki Kosugi1, Masayuki

Shimase1, Yuta Katayama2, Yasuyuki Nakayama1, and Junji Watari1

ABSTRACT

J. Inst. Brew. 116(3), 251–260, 2010

The behaviour of hop-derived monoterpene alcohols during fermentation by lager yeast was previously investigated in this laboratory. It was suggested that the concentration of geraniol and β-citronellol in the finished beer increased depending on the initial concentration of geraniol in the wort. In addition, an addi-tive effect among linalool, geraniol and β-citronellol was found and 5 ug/L of geraniol and β-citronellol was enough for this effect. In this paper, conditions regarding the enrichment of the initial concentration of geraniol in the wort were investigated. From the screening of various hop cultivars, Citra hop was se-lected as a geraniol-rich cultivar. In addition, it was observed that coriander seed, which can be used in beer production as a flavourant, contained not only linalool but also geraniol at high levels. The use of Citra hop or coriander seed was effective for enriching the concentration of geraniol and β-citronellol in the finished beers. In the Citra beer and in the coriander beer, the content of linalool was excess in comparison with the content of geraniol and β-citronellol. Therefore, the synergy of geraniol and β-citronellol, under coexistence of excess linalool, was ex-amined. It was found that the flavour impression of excess li-nalool became more fruity and citrus by coexistence with gera-niol and β-citronellol and that the coexistence of all three monoterpene alcohols was effective for this synergy. The flavour characteristics of the Citra and coriander beer and the impor-tance of geraniol metabolism for a citrus flavour in beer are discussed.

Key words: β-citronellol, beers, biotransformation, Citra, cori-ander, flavour, geraniol, hops, linalool, monoterpene alcohols, synergy.

INTRODUCTION Monoterpene alcohols, including linalool and geraniol,

have very floral aromas and contribute to the aromas of various foods and beverages, such as fruits, juices, wines and beers. In the field of beer investigation, it is well known that terpenoids in finished beers are mainly de-rived from hops (Humulus lupulus L.). Among the five monoterpene alcohols (linalool, α-terpineol, β-citronellol, nerol and geraniol), linalool is found in various beers and is regarded as an important factor for a hop-derived beer flavour4,14,20. Geraniol however is considered to be more cultivar-specific than linalool18,23. Several researchers have pointed out that Cascade, a U.S. hop cultivar, contains not only linalool but also geraniol in the hop cone, pellet, hop oil and finished beer3,18,23,24. Wines also contain monoter-pene alcohols as major flavour compounds, however the monoterpene alcohols in wines are mainly derived from wine grapes (Vitis vinifera L.). It is well known that li-nalool is widely contained in wines made from various cultivars of wine grapes. Linalool in particular is regarded as one of the key compounds contributing to the cultivar-specific flavours of Muscat wine and Riesling wine. As with hops, geraniol is considered to be more cultivar-spe-cific than linalool and is found in wines made from cer-tain cultivars, such as Muscat and Gewürztraminer1.

In the field of beer flavour chemistry, several research-ers have proposed that geraniol is biotransformed to β-citronellol during fermentation13,20. Recently, King and Dickinson8,9 reported the biotransformation of monoter-pene alcohols by yeast metabolism. They proposed me-tabolism cascades of monoterpene alcohols in various yeasts, including wine, lager and ale yeasts, as a result of systematic model fermentations containing each monoter-pene alcohol. Fig. 1 shows the metabolism cascade of monoterpene alcohols by lager and ale yeast9. Geraniol can be mainly transformed to β-citronellol and adjunc-tively to linalool. Nerol can be converted to linalool and α-terpineol. A part of linalool can be cyclised to α-ter-pineol. The biotransformation of geraniol to β-citronellol, in particular, was observed to be rapid within the first 2-4 days of a model fermentation. A decrease in geraniol and a corresponding increase in β-citronellol occurred during this period8,9.

1 Value Creation Department, Sapporo Breweries Ltd., 10 Oka-tohme, Yaizu, Shizuoka, 425-0013, Japan.

2 Frontier Laboratories of Value Creation, Sapporo Breweries Ltd.,10 Okatohme, Yaizu, Shizuoka, 425-0013, Japan.

3 Bioresources Research and Development Department, SapporoBreweries Ltd., 3-5-25 Kamifurano-cho Motomachi, Sorachi-gun, Hokkaido, 071-0551, Japan.

* Corresponding author. E-mail: [email protected]

Publication no. G-2010-1020-1081 © 2010 The Institute of Brewing & Distilling

252 JOURNAL OF THE INSTITUTE OF BREWING

This laboratory has previously reported on the bio-transformation of monoterpene alcohols during the fer-mentation of hopped beer using various hop cultivars27. Geraniol was observed to decrease during the first 3 days. β-Citronellol was almost absent in the wort and gradually increased during the total fermentation period. The con-centrations of geraniol and β-citronellol in the finished beer increased depending on the initial concentration of geraniol in the wort. In the fermentation of hopped beer, the gradual increase in β-citronellol did not correspond to the fast decrease of geraniol. This increase of β-citronellol might be partly explained by the occurrence of a glycosi-dically bound flavour precursor and glucoside hydrolase activity secreted from the lager yeast.

The flavour characteristics of monoterpene alcohols have also been reported27. Table I lists the olfactory de-scriptions of monoterpene alcohols and their group thresholds as determined by the sensory panellists. The thresholds of α-terpineol and nerol were much higher than

those of linalool, geraniol and β-citronellol. In test brewed products, the concentrations of α-terpineol and nerol in the hopped beers were almost lower than those of the other three monoterpene alcohols. Therefore, it was speculated that α-terpineol and nerol might not contribute significantly to hopped beer flavour. Furthermore, the flavour characteristics of linalool, geraniol and β-citronel-lol were examined and an additive effect was found among these three monoterpene alcohols. Only with 5 ug/L of geraniol and β-citronellol was there enough to produce this effect. It was thus suggested that not only linalool, but also geraniol and β-citronellol may contribute to a hopped beer flavour at lower levels, i.e., where the OAVs (OAV: ratio of concentration to odour threshold) of these compounds are below 1.0.

In this paper, the behaviour of monoterpene alcohols during fermentation under geraniol enriched conditions was examined using geraniol rich hops or coriander seeds. Changes in flavour impression by the synergy of geraniol

Table I. Olfactory descriptions and perception thresholds of the monoterpene alcohols (μg/L) in a model solution (5% v/v ethanol, carbonated) and a commercial beer (as previously reported in reference 27).

Olfactory perception thresholda

Reference compound Olfactory description Model solutionb Beerc

Linalool (µg/L) lavender 3d 5d

α-terpineol (µg/L) lilac 450d - β-citronellol (µg/L) lemon, lime 9d 8d Nerol (µg/L) rose, citrus 80 - Geraniol (µg/L) rose 7 6 a Flavour threshold determined by 10–13 panellists. b Model carbonated dilute ethanol solution (5% v/v ethanol, carbonated). c Japanese commercial beer. d Determined using racemic mixture.

Fig. 1. Metabolism cascade of monoterpene alcohols by lager and ale yeast proposed by King andDickinson8,9. The asterisk indicates a chiral centre (This figure was previously shown in reference27).

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and β-citronellol under coexistence with excess linalool were evaluated.

MATERIALS AND METHODS Raw materials

Cascade, Citra, Millennium and Nugget hops were grown in the U.S. Hallertauer Tradition (HHT) and Hallertauer Magnum (HHM) hops were grown in Ger-many. All hops were harvested in 2007. Coriander seed was grown and harvested in Canada in 2006.

Hop extraction with hot water

For analysis of the flavour compounds, all hops were extracted with hot water. Hop pellets (~3–5 g, see Table II) were added to 200 mL of water and autoclaved at 105°C for 5 min. After cooling on ice-water, the mixture was passed through a filter paper and the eluant obtained was the hop water extract. Hop dosage was adjusted ac-cording to the α acid content2 of each hop.

Pilot-scale brewing

Beers and Happoshu (a Japanese low-tax beer using less than 24% malt) were produced using Citra hop or coriander seed and the same recipe as the standard method of the Production & Technology Development Centre, Sapporo Breweries, Ltd. Briefly, the wort was prepared using commercially available malts, adjuncts and hops in a 400- or 5,000-L scale pilot apparatus. The worts of the ‘all malt’ beer were produced with only malt. The Happoshu worts contained 24% malt, 20% barley and 56% syrups. Boiling periods were 90 min for beer and 70 min for Happoshu. For production of the Citra beers, hops (Citra hop) were added at the beginning of the boil (~10% of total dosage) and at 5 min before the end of the boil (~90% of total dosage). For production of the coriander beers, hops (the HHT hop) were added at the beginning of the boil (50% of total dosage) and at 5 min before the end of the boil (50% of total dosage). In addition, coriander seed and orange peel were added 5 min before the end of the boil. After cooling, lager yeast (Saccharomyces pas-torianus, brewery collected, 15.0 × 106 – 30.0 × 106 cells/mL) was added to the cooled wort. The temperature of the fermentation was maintained at 10–15°C. The fer-mented wort was transferred to another storage tank under a CO2 atmosphere and maturation was carried out at 13–15°C for 6–8 days and then at 0°C for 2–3 weeks. Filtra-tion and bottling were carried out using pilot-scale equip-ment under anti-oxidative conditions.

Chemicals

Linalool (>98%, racemic mixture), α-terpineol (>95%, racemic mixture), nerol (>98%) and β-citronellol (>92%, racemic mixture) were purchased from the Tokyo Chemi-cal Industry Co., Ltd. (Tokyo, Japan). Geraniol (98%) was purchased from Aldrich Chemical Co. Inc. (Milwaukee, USA).

Quantification of monoterpene alcohols by gas chromatography-mass spectrometry (GC-MS)

GC-MS analysis was carried out using a 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA). The carrier gas was helium with a column-head pressure of 15 psi and flow rate of 1.8 mL/min. The detector was a mass spectrometer (MS 5973, Agilent Technologies) func-tioning in the EI mode (70 eV) and was connected to the GC by a transfer line heated to 280°C. For analysis of raw hops, 20 mg of ground hop was placed directly into a 20 mL glass vial. For analysis of the hop water extract, wort, fermenting beer and finished beer, an 8 mL aliquot of each sample was placed into a 20 mL glass vial along with 3 g of sodium chloride at 0°C. For the analysis of raw coriander seed, a 200 mg aliquot of ground coriander seed was placed directly into a 20 mL glass vial. The vial, including the sample, was sealed with a magnet cap. The vial was pre-incubated with stirring at 40°C for 15 min using a Combi-PAL autosampler (CTC Analytics, Zwin-gen, Switzerland). After pre-incubation, a SPME fibre [PDMS (polydimethylsiloxane), 100 µm film thickness, Supelco, Bellefonte, PA, USA] was inserted into the head space of the vial and adsorption was carried out for 15 min. After adsorption, the SPME fibre was injected into a splitless injector (260°C, purge time 3 min, purge flow 20 mL/min) at an oven temperature of 50°C onto an HP-1MS capillary column (Agilent Technologies, 30 m, 0.25 mm internal diameter (i.d.), 1.0 µm film thickness). For all analyses, the temperature program was as follows: 50°C for 1 min, raised at 5°C/min to 250°C, followed by a 1 min isotherm. The monoterpene alcohols (linalool, α-ter-pineol, β-citronellol, nerol and geraniol) were quantified in the SIM mode, with selection of the following ions: m/z 93 (for α-terpineol, nerol and geraniol) and 109 (for linalool and β-citronellol). Calibration curves were deter-mined using water (including 5% ethanol) containing the monoterpene alcohols at final concentrations ranging from 0 to 10 µg/L. All calibrations produced a linear re-sponse with an R2 value >0.98 over the concentration range analyzed.

Sensory evaluation of synergy among monoterpene alcohols

Each sensory evaluation was performed by 10-13 well-trained panellists. The change of flavour characters by synergy among monoterpene alcohols was assessed in a model solution (5% v/v ethanol, carbonated), as follows. For simulation of the composition of the three monoter-pene alcohols in the Citra beer, a control solution contain-ing 70 µg/L of linalool and test solutions containing the same concentration of linalool together with 15 µg/L of geraniol and/or 20 µg/L of β-citronellol were prepared.

Table II. Conditions of the hop water-extraction.

Hop cultivar BUa α-Acid (%)a Hop dosageb (g/200 mL)

HHTc 36.0 04.5 4.62 HHMd 101 11.9 1.75 Nugget 93.9 12.7 1.64 Millennium 104 14.5 1.43 Cascade 45.6 05.3 3.92 Citra 89.7 12.1 1.72 a Measured according to Analytica-EBC2. b Hop dosage for hop water extraction. c Hallertauer Tradition. d Hallertauer Magnum.


For simulation of the composition of three monoterpene alcohols in the coriander beer, a control solution contain-ing 1,000 µg/L of linalool and test solutions containing the same concentration of linalool together with 25 µg/L of geraniol and/or 20 µg/L of β-citronellol were prepared. A 50 mL aliquot of each sample solution was presented in a plastic cup and the five flavour characters (flowery, fruity, citrus, green and artificial) were scored from 0 (no flavour) to 3 (strong flavour) in intervals of 1.0.

RESULTS Screening of geraniol-rich hop

In this work, the effect of geraniol metabolism on beer flavour was investigated in detail. It had already been re-ported that the content of geraniol and β-citronellol in finished beer could be increased by enriching the initial geraniol content in the wort27. In beer production, hops

(Humulus lupulus L.) are the main source of geraniol. Several researchers have pointed out that geraniol is more cultivar-specific than linalool18,23. Therefore, a screening of geraniol rich hops, by comparing the composition of the monoterpene alcohols (linalool, α-terpineol, β-citronellol, nerol and geraniol), was conducted. Six hop cultivars, Hallertauer Tradition (HHT) a traditional German aroma hop, Hallertauer Magnum (HHM) a major German high-alpha hop, Cascade a typical U.S. aroma hop, Nugget and Millennium U.S. high-alpha hops and Citra a newly bred dual purpose U.S. hop, were compared. The content of monoterpene alcohols in the ground hops was regarded as the content before the boil. The content in the hot water hop extract (autoclaved at 105°C for 5 min; this condition corresponded to that of late-hopping) was regarded as the content after the boil. For comparison, these numbers are presented as µg/g of hop.

Fig. 2. Composition of monoterpene alcohols (µg/g of hop) before and after the boil (data of the HHT hop was previously reported in reference 27).

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Fig. 2 shows the composition of the five monoterpene alcohols in the hops before and after the boil. Linalool was found in all of the cultivars at a significant level be-fore and after the boil. Geraniol was contained in all of the cultivars at quantifiable or significant levels. The com-pounds α-terpineol, β-citronellol and nerol were found in trace or low levels. As described in previous re-ports3,18,23,24, Cascade hops contained not only linalool, but also geraniol at significant levels. Of the six cultivars, Citra hop contained the largest amount of geraniol. Citra hop was bred in the U.S. and released in 2007 by the Hop Breeding Company. Breeders have reported that this hop imparts a cultivar-specific flavour to the finished beer somewhat reminiscent of grapefruit, passion fruit, goose-

berry, lime and lychee19. This cultivar was thought to be suitable for evaluating the effect of geraniol metabolism on beer flavour, because of the large amount of geraniol in this hop.

Behaviour of monoterpene alcohols during fermentation of beers made with Citra hop

Three test beers were brewed with the pilot brewing apparatus using Citra hops. At 5 min before the end of the boil, 0.8 g/L of Citra hop was added for the “all malt” beer, 0.4 g/L for the “Happoshu 1” and 0.8 g/L for the “Happoshu 2”. Fig. 3 shows the behaviour of linalool, β-citronellol and geraniol during the fermentation of these beers. The content of linalool in the finished beers was around half of the concentration in the worts (Fig. 3A). The compound β-citronellol was present at trace levels in the worts and increased continuously up to ~10–20 µg/L during the total fermentation period (Fig. 3B). In the worts, the geraniol content was at a significant level (~60–120 µg/L) and a large decrease in the geraniol content occurred during the first 3 days fermentation, as in the previous report27. However, ~10–20 µg/L of geraniol re-mained in the beers (Fig. 3C). The content of the β-citro-nellol generated and geraniol retained in the all malt beer and the Happoshu 2, late-hopped with 0.8 g/L of the Citra hop, was twice that in Happoshu 1, late-hopped with 0.4 g/L of this hop. This confirmed that the content of β-citro-nellol and geraniol in the finished beer could be enriched, depending on the initial content of geraniol in the wort.

Behaviour of monoterpene alcohols during fermentation of beers produced with coriander seed

The next focus was on coriander seed as a source of geraniol. Coriander seeds have frequently been used as a flavourant, together with hops, for certain traditional beers such as Belgian style white beer. Coriander (Coriandrum sativum L.) is an annual herb in the Apiaceae family. The fresh or dried leaves are commonly used as an herb and the dried seeds are used primarily as a spice in cooking. It is well-known that linalool is the main component in cori-ander essential oil. Linalool is over 70% of the total oil content. Several other terpenoids are also contained in coriander oil such as geraniol, geranyl acetate, α-pinene and camphor5,21. Table III shows a comparison of the con-centration of the monoterpene alcohols in some Canadian commercial craft beers, all of which were produced using coriander seed, together with hops, according to the recipe of a Belgian style beer. In these beers, the concentration of linalool, β-citronellol and geraniol was as follows: li-nalool 393–906 µg/L; β-citronellol 14.6–49.3 µg/L; and geraniol 43.8–89.6 µg/L. From these results, it was as-sumed that the worts made with coriander seed contained

Fig. 3. Comparison of monoterpene alcohols (µg/L) during fer-mentation of the Citra beer (Ferm – fermentation; d – days; w –week). (A) linalool; (B) β-citronellol; (C) geraniol. Hop dosageat 5 min before the end of the boil was 0.8 g/L for “All malt”, 0.4 g/L for “Happoshu 1” and 0.8 g/L for “Happoshu 2”.

Table III. Comparison of the concentration of monoterpene alcohols (µg/L) in a number of commercial Canadian craft beers.

Brand A Brand B Brand C Brand D Brand E

Linalool (µg/L) 444 554 697 906 393 α-terpineol (µg/L) 44.6 65.8 92.5 155 76.1 β-citronellol (µg/L) 14.6 20.9 36.9 49.3 34.0 Nerol (µg/L) 7.8 11.6 15.4 23.8 12.6 Geraniol (µg/L) 55.3 54.8 61.5 89.6 43.8


very large amounts of linalool and geraniol, and that a large amount of β-citronellol was biotransformed from geraniol during fermentation.

In order to confirm the behaviour of the monoterpene alcohols during fermentation of the beers produced with coriander seed, two beers were brewed with our pilot brewing apparatus using coriander seed. At 5 min before the end of the boil, 0.5 g/L of the coriander seed was added for “coriander 1” beer, 0.75 g/L for “coriander 2” beer. Although 0.4 g/L of the HHT hop and 0.2 g/L of orange peel were also added together with the coriander seed, the geraniol content due to these other materials was at trace levels. The geraniol in these worts was derived primarily from the coriander seed. Fig. 4 shows the be-haviour of linalool, β-citronellol and geraniol during the fermentation of these beers. The content of linalool in the finished beers was approximately two thirds of the con-tent in the worts (Fig. 4A). The β-citronellol was present at a low level (~5 µg/L) in the worts and increased con-tinuously up to 20–30 µg/L during the total fermentation period (Fig. 4B). The geraniol content in the wort was at a high level (~130–170 µg/L) and a large decrease in gera-niol was observed during the first 3 days fermentation, as in the Citra beers. However, ~25–35 µg/L of geraniol re-

mained in the finished beers (Fig. 4C). This confirmed that using not only the geraniol-rich hop but also the cori-ander seed could enrich the content of geraniol and β-citronellol in the finished beer.

Synergy of geraniol and β-citronellol under coexistence with excess linalool

The Citra beer had a characteristic citrus and green aroma. The coriander beer had a very floral aroma with a slight citrus impression. It was assumed that geraniol and β-citronellol might contribute to the citrus flavour of these beers. This laboratory has previously published that there is an additive effect with linalool, geraniol and β-citronel-lol, and that this effect might contribute to the citrus fla-vour of hopped beer27. However, in the Citra beer and in the coriander beer, the content of the linalool was in ex-cess in comparison with geraniol and β-citronellol (Fig. 3 and 4). Table IV shows the composition of these three monoterpene alcohols in the Citra beer (“all malt” beer in Fig. 3) and the coriander beer (“coriander 1” beer in Fig. 4). Under these conditions, the strong flavour of linalool might antagonize the flavours of the geraniol and β-citro-nellol in spite of an additive effect among these monoter-pene alcohols. On the other hand, it has been reported that several flavour compounds can affect a total flavour im-pression, despite small amounts below their respective thresholds, by coexistence with certain key flavour com-pounds, which have a very low threshold and could func-tion as a flavour enhancer25,26. It was speculated that excess linalool could antagonize the flavours of geraniol and β-citronellol or function with these compounds as a flavour enhancer. Therefore, a test was conducted to assess changes of flavour character by synergy of these three monoterpene alcohols. Figs. 5 and 6 show the five flavour characters (flowery, fruity, citrus, green and artificial) of model solutions, evaluated by the panellists. These model solutions were designed to simulate the composition of the three monoterpene alcohols in the Citra and coriander beer.

For simulation of the composition of the three mono-terpene alcohols in the Citra beer (“all malt” beer in Fig. 3), a control solution containing 70 µg/L of linalool and test solutions containing the same concentration of li-nalool together with 15 µg/L of geraniol and/or 20 µg/L of β-citronellol were prepared (Fig. 5). With the addition of 15 µg/L of geraniol, the average scores of “citrus” and “green” characters increased slightly in comparison with the control solution. The spider chart of the test solution was almost similar shape to that of the control solution (Fig. 5A and B). With the addition of 20 µg/L of β-citro-nellol, the average scores of the “flowery” and “fruity”

Fig. 4. Comparison of monoterpene alcohols (µg/L) during fer-mentation of a coriander beer. Coriander dosage at 5 min beforethe end of the boil was 0.5 g/L for “Coriander 1” and 0.75 g/Lfor “Coriander 2”.

Table IV. Comparison of the composition of the monoterpene alcohols linalool, geraniol and β-citronellol in two test beers.

Citra beera Coriander beerb

Linalool (µg/L) 69.0 949 β-citronellol (µg/L) 14.0 24.0 Geraniol (µg/L) 22.6 20.3 a Beer was brewed using 0.4 g/L of Citra hop at 5 min before the end of

the boil (“all malt” beer in Fig. 3). b Beer was brewed using 0.5 g/L coriander seed at 5 min before the end

of the boil (“coriander 1” in Fig. 4).

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Fig. 5. Flavour profile of model solutions simulating the composition of three monoterpene alcohols in a Citra beer.

Fig. 6. Flavour profile of model solutions simulating the composition of three monoterpene alcohols in a coriander beer.


character decreased and “citrus”, “green” and “artificial” increased in comparison with the control solution (Fig. 5A and C). Among all of the flavour characters, the “green” character showed the most change. With the addition of 15 µg/L of geraniol and 20 µg/L of β-citronellol, the average score of the “flowery” character decreased slightly and the average scores of the “fruity”, “citrus”, “green” and “artificial” character increased in comparison to the control solution (Fig. 5A and D).

For simulation of the composition of the three mono-terpene alcohols in the coriander beer (“coriander 1” beer in Fig. 4), a control solution containing 1,000 µg/L of linalool and test solutions containing the same concentra-tion of linalool together with 25 µg/L of geraniol and/or 20 µg/L of β-citronellol were prepared (Fig. 6). With the addition of 25 µg/L of geraniol, the average score of the “green” and “artificial” character increased slightly in comparison with the control solution. The spider chart of the test solution was almost similar shape to that of the control solution (Fig. 6A and B). However, with the addi-tion of 20 µg/L of β-citronellol, the average score of the “fruity” character increased. The average scores of the other four characters were similar to the levels in the con-trol solution (Fig. 6A and C). With the addition of 25 µg/L of geraniol and 20 µg/L of β-citronellol, the average scores of all five of the characters increased in compari-son to the control solution (Fig. 6A and D).

This suggests that geraniol and β-citronellol can affect the total flavour impression under a condition coexisting with excess linalool, and that coexistence of geraniol and β-citronellol can effectively change flavour characters. The “citrus” character of the test solutions was in particu-lar maximized by coexistence with the three monoterpene alcohols. Therefore, it is proposed that the synergy of these three monoterpene alcohols can contribute to a part of the citrus flavour in beer.

DISCUSSION Flavour characteristics of beers made with Citra hop

Hop breeders in the U.S. have reported that Citra hop imparts a cultivar-specific flavour to the finished beer and that this specific flavour is somewhat reminiscent of grapefruit, passion fruit, gooseberry, lime, and lychee19. However, there have been no studies on key flavour com-pounds contributing to the cultivar-specific flavour of the beers made with Citra hops.

In this study, it was found that the Citra hop contains both linalool and geraniol at very high levels, and that the finished Citra beer contains not only linalool and geraniol but also β-citronellol, which is biotransformed from gera-niol during fermentation. As a result of the sensory eval-uations, a flavour description of standard β-citronellol is citrus and slightly green, such as a young lemon or a lime (Table I). Therefore, it was assumed that β-citronellol might contribute to the lime-like flavour of the Citra beer by coexisting with the other two monoterpene alcohols. From the test brews it was shown that the finished Citra beers contained excess linalool (~70 µg/L) and relatively small amounts of geraniol and β-citronellol (~15–20 µg/L). Tests were conducted to evaluate changes in the

five flavour characters (flowery, fruity, citrus, green and artificial) by synergy among three monoterpene alcohols by the sensory evaluation of model solutions that simu-lated the composition of the three monoterpene alcohols in the Citra beer. It was observed that 15 µg/L of geraniol and 20 µg/L of β-citronellol could enhance the “fruity”, “citrus”, “green” and “artificial” character by coexisting with 70 µg/L of linalool (Fig. 5). Therefore, it was pro-posed that synergy of these monoterpene alcohols might contribute to the lime-like citrus flavour of the beers brewed using Citra hops.

In addition, there was a focus on the lychee-like flavour derived from the Citra hop19. Lychee (Litchi chinesis Sonn.) is a subtropical fruit belonging to the Sapindaceae family and is grown primarily in China. This fruit has a charac-teristic rose-floral and citrus-like aroma. It has been re-ported that the key flavour compounds of lychee fruit are cis-rose oxide, β-damascenone, furaneol, linalool, geraniol, β-citronellol and some esters, such as isobutyl acetate, ethyl isobutyrate, ethyl isohexanoate, isoamyl acetate15,16,30. Among these key compounds, cis-rose oxide has shown the highest OAV value15,16. It is also well-known that Gewürztraminer wine has a very unique aroma, reminis-cent of lychee. Several common key compounds are found in both Gewürztraminer wine and lychee fruit16, for ex-ample cis-rose oxide, linalool, geraniol and β-citronellol. Vaudano et al.29 reported that the biotransformation of geraniol to β-citronellol occurs during the fermentation of geraniol-rich grape cultivars, such as Gewürztraminer. In the test brewed Citra beer, cis-rose oxide was not detected (data not shown) and the monoterpene alcohols were con-tained at significant levels (Fig. 3). Therefore, it was as-sumed that the lychee-like character derived from the Citra hop, might be partly explained by the occurrence of the monoterpene alcohols, especially geraniol for a rose-floral character and β-citronellol for a citrus character.

As described above, hop breeders have reported that the Citra hop gives a cultivar-specific flavour, which was somewhat reminiscent of grapefruit, passion fruit, goose-berry, lime and lychee to the finished beer19. In the field of wine flavour investigation, it is well known that several volatile thiols (3-sulfanylhexan-1-ol and 3-sulfanylhexyl acetate) have a grapefruit-like and/or passion fruit-like flavour28. It has also been reported that certain volatile thiols with a grapefruit-like flavour (3-sulfanyl-4-methyl-pentan-1-ol and 3-sulfanyl-4-methylpentyl acetate) occur in beers made from Nelson Sauvin hops25,26. Therefore, the grapefruit-like flavour of the Citra beer might be derived from such volatile thiols. However, the grapefruit-like fla-vour derived from volatile thiols is very characteristic. It is thought that the citrus flavour of the beer could not be com-pletely explained only by these volatile thiols. In this study, Citra hop was selected as a geraniol-rich hop and the main focus was on the lime-like flavour of the Citra beer. The flavour of the Citra beer is very complicated and it is ex-pected that various other flavour compounds may well contribute to the overall flavour impression of Citra beers.

Flavour characteristics of beers made with coriander seed

Coriander seeds have been used as a flavourant, to-gether with hops, for certain traditional beers, such as

VOL. 116, NO. 3, 2010 259

Belgian style white beer. It is well known that these types of beers are produced primarily using barley malt, wheat, hops, orange peel and coriander seed, and that such beers have a very floral and slight citrus aroma and a slight sour taste. It has traditionally been considered that coriander seed contributes to the floral aroma, orange peel to the citrus aroma and wheat to the sour taste. However, there have been no reported studies on the behaviour of mono-terpene alcohols during the fermentation of beers pro-duced with coriander seed, in particular from the view-point of biotransformation by brewing yeasts.

According to previous papers15,16,30, it was expected that the coriander seed would be a source not only of lina-lool, but also of geraniol. It was confirmed that the worts produced with coriander seed contained large amounts of linalool and geraniol, and that a part of the geraniol was biotransformed to β-citronellol during the fermentation process (Fig. 4). The test brews showed that the finished coriander beers contained excess linalool (~1,000–1,200 µg/L) and relatively small amounts of geraniol and β-citronellol (~20–30 µg/L). The effects of such amounts of geraniol and β-citronellol on the total flavour impression, under coexistence with excess linalool, were assessed by a sensory evaluation of model solutions simulating the com-position of the three monoterpene alcohols in the corian-der beer. As a result, it is suggested that 25 µg/L of gera-niol and 20 µg/L of β-citronellol could enhance the “fruity”, “citrus” and “green” characters while coexisting with 1,000 µg/L of linalool (Fig. 6). Therefore, it is pro-posed that not only orange peel, but also coriander seed, may contribute to the citrus flavour of the beers produced using coriander seed.

Importance of geraniol metabolism for citrus flavour in beer

Recently, techniques of gas chromatography-olfac-tometry (GC-O) have been developed and applied in fla-vour chemistry. In the field of beer investigation, several researchers have reported analyses of hop-derived flavour compounds by GC-O3,11,14,23. However, little has been re-ported on β-citronellol in these papers. As described above, geraniol has been considered to be more cultivar-specific than linalool18,23, and the β-citronellol content de-pends on the initial geraniol content in the wort27. There-fore, it could be difficult to detect β-citronellol in beers produced using geraniol-poor hops (i.e., German aroma hops)23,27. In addition, Kishimoto et al.10 have reported that using extraction with solvent they could not detect β-citronellol in Japanese beers, while with stir bar-sorptive extraction (SBSE) they could detect this compound in the same beers. Although several researchers have reported the results of their GC-O analyses preparing volatile com-pounds by using extraction with solvent3,11,14,23, they have not mentioned the detection of β-citronellol. In this study, solid phase micro extraction (SPME) was used as the preparation method for GC-MS analysis. The SPME fibre, polydimethylsiloxane (PDMS) was used as an absorbent for volatile compounds, as well as in the SBSE stir bar. Therefore, SPME might be a suitable method for the de-tection of β-citronellol. It was confirmed that GC-O with the SPME fibre could detect β-citronellol in the Citra beer (data not shown).

It was previously reported that there was an additive ef-fect among linalool, geraniol and β-citronellol, and that only 5 ug/L of geraniol and β-citronellol were enough for this effect27. In this paper, it has been shown that geraniol and β-citronellol can affect the total flavour impression under coexistence with excess linalool, and that the syn-ergy of these three monoterpene alcohols can contribute to a citrus flavour in beer. The spider charts of the test solution, containing linalool and geraniol, were almost similar in shape to those of the corresponding control solution containing only linalool (Figs. 5A, 5B, 6A and 6B). Therefore, it was thought that the effect of geraniol on total flavour impression was restricted, because the flavours of linalool and geraniol are both flowery. On the other hand, the spider charts of the test solution, contain-ing linalool and β-citronellol, characteristically changed in comparison with those of the corresponding control solution (Figs. 5A, 5C, 6A and 6C). The changes in the spider charts became the largest by coexistence with the three monoterpene alcohols (Figs. 5A, 5D, 6A and 6D). These results suggest an importance of β-citronellol in the hop-derived citrus flavour of beer. It was found that there was little β-citronellol in raw hops (Fig. 2) and that the generation of β-citronellol depended on geraniol metabo-lism by the yeast (Figs. 3 and 4). Therefore, it was con-cluded that all of the geraniol-rich conditions (using gera-niol-rich hop or coriander seed), geraniol metabolism by yeast and occurrence of three monoterpene alcohols (li-nalool, geraniol and β-citronellol) might contribute to enhancing the hop-derived citrus flavour of beer.

In this study, a commercial racemic mixture of linalool and β-citronellol was used for sensory evaluation. The stereochemistry of these compounds has recently been investigated. From a flavour chemistry viewpoint, the threshold of (R)-linalool is lower than the (S)-isomer and the racemic mixture12,22. Kaltner et al.7 reported that (R)-linalool dominates in various raw hops and that the ratio of the (R)-isomer was almost above 90%. Racemisation of linalool was observed during the brewing and beer staling process. In kettle-hopped beer, R/S ratios have been near to the racemate. In late-hopped and dry-hopped beers, (R)-linalool dominated7,12,22. It has also been reported that (S)-linalool dominates in coriander seed17. In addition, Gramatica et al.6 reported a stereospecific reduction of geraniol to (R)-β-citronellol by Saccharomyces cerevisiae. From these previous reports, the enantiomeric ratio of linalool and β-citronellol in beer would be expected to change depending on the flavourant, the brewing process and the beer staling process. The effect of the R/S ratios on the synergy among monoterpene alcohols should be revealed in the future.

ACKNOWLEDGEMENTS

We acknowledge Nobuaki Yamaura, Junji Takayanagi, Takumi Shibata and Reika Miyamoto at the Frontier Laborato-ries of Value Creation for technical assistance, all panellists at the Value Creation Department and the Frontier Laboratories of Value Creation for their sensory work, and Akira Inaba at the Bioresources Research & Development Department, Koichi Toyoshima and Kazutoshi Ito at the Frontier Laboratories of Value Creation for their kind help.


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(Manuscript accepted for publication July 2010)

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Bio Transformation of Geraniol