Identification and Characterization of Geranic acid as Unique Flavor

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Chemistry and Biology of Aroma and Taste

Identification and Characterization of Geranic acid as Unique Flavor Compound of Hops (Humulus lupulus L.) Variety Sorachi Ace Ayako Sanekata, Atsushi Tanigawa, Kiyoshi Takoi, Yasuyuki Nakayama, and Youichi Tsuchiya J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04395 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Journal of Agricultural and Food Chemistry

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Title

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Identification and Characterization of Geranic acid as

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Unique Flavor Compound of Hops (Humulus lupulus L.)

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Variety Sorachi Ace

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Authorship

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Ayako Sanekata1,*†, Atsushi Tanigawa2, Kiyoshi Takoi2†, Yasuyuki Nakayama2, Youichi Tsuchiya1

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1

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ka, 425-0013 Japan

Frontier Laboratories for Value Creation, SAPPORO HOLDINGS LTD., 10 Okatome, Yaizu, Shizuo-

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2

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Shizuoka, 425-0013 Japan

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*Corresponding author: TEL. +81-54-629-7980

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†Authors contributed equally to this work.

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E-mail: [email protected]

Product & Technology Innovation Department, SAPPORO BREWERIES LTD., 10 Okatome, Yaizu,

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1 ACS Paragon Plus Environment


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Abstract

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Hops are natural ingredients used to impart bitterness and flavor to beer. Recently, new varieties of hops

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have attracted global research attention. The Sorachi Ace variety, especially, interests many craft brew-

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ers. This hop imparts characteristic varietal aromas, including woody, pine-like, citrus, dill-like, and

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lemongrass-like, to finished beers. Here, we investigated specific flavor compounds derived from

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Sorachi

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olfactometry/mass spectrometry and head space-solid phase microextraction-gas chromatography-mass

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spectrometry. The results showed that a unique volatile compound, geranic acid, was present at a signif-

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icant level only in the test beer brewed with the Sorachi Ace hop. Furthermore, sensory evaluation tech-

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niques revealed that geranic acid has very unique characteristics. This compound is not odor-active but

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functions as an enhancer for hop-derived terpenoids at subthreshold levels.

Ace

using

selectable

one-dimensional

or

two-dimensional

gas

chromatography-

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Keywords

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Sorachi Ace, beer, hops, varietal aroma, GC-O, HS-SPME-GC-MS, geranic acid, geraniol, linalool

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ACS Paragon Plus Environment

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Introduction

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In recent years, there has been increased production and consumption of craft beers worldwide. India

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Pale Ale (IPA) is a very popular beer style in the craft-beer market. In general, IPA is strongly dry-

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hopped as opposed to other styles of craft beers. Craft brewers often use new hop varieties with charac-

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teristic flavors, the so-called “flavor hops,” for IPA beers. Flavor hops are a new type of hops1, 2 and are

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also called as “special flavor hops” and “impact hops”3. Each variety of flavor hops imparts a character-

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istic citrus-like and/or exotic fruit-like (tropical) aroma to the finished beer. Such hops are being bred

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and used widely for craft beers1, 4-8. For example, consider Nelson Sauvin, Citra, and Mosaic, which are

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typical flavor hop varieties. Nelson Sauvin has white wine-like aroma characteristics and fruitiness with

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fresh crushed gooseberry and grape-infused flavors, while the aroma imparted by Citra is like grapefruit,

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melon, lime, and passion fruit. The varietal aroma of Mosaic, on the other hand, is blueberry, tangerine,

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papaya, rose, blossoms, grass, and mango. Takoi et al. described the contribution of monoterpene alco-

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hols and volatile thiols to hop-derived varietal aromas, the behavior of these compounds during beer

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production, and the mechanism of varietal aroma development based on the synergy between the flavor

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compounds in several hop varieties, such as Bravo, Cascade, Citra, Mosaic, and Nelson Sauvin9-17.

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Sorachi Ace hop is gaining popularity among craft brewers, and it is used to impart pine-like, woody,

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dill-like, citrus, lemongrass-like flavors owing to its unique varietal aroma. Although Sorachi Ace is

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now mainly grown in the U.S., it was originally bred in Sorachi-gun, Hokkaido, Japan, around 30 years

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ago by SAPPORO BREWERIES LTD., and named after its place of origin. In 1975, the initial breeding

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process was carried out using a Brewer’s Gold female hop plant and a Saaz male hop plant. From these

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crosses, the 70K-SH6 female hop plant was selected and crossed with a Beikei 2 male hop plant. Finally,

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Sorachi Ace was produced (Figure 1) and the variety was registered in 1984 in Japan18. Subsequently,

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this hop was brought to the U.S. in 1994 by a Japanese hop breeder and is now grown on commercial-

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scale farms in the states of Washington and Oregon. In the 2010s, Sorachi Ace gained much attention

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because of its very characteristic varietal aroma and was widely used for craft beers across the U.S.,

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Belgium, Japan, and several other countries.

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Several researchers investigated the composition of hop-derived flavor compounds in various hops and

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beers, including the Sorachi Ace hop and beer17, 19-22. However, the key compounds contributing to the

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varietal aroma of Sorachi Ace have not been fully investigated. In this study, we tried to identify the va-

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riety-specific flavor compounds derived from the Sorachi Ace hop using selectable one-dimensional or

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two-dimensional gas chromatography-olfactometry/mass spectrometry (1D or 2D GC-O/MS) and head-

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space-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). We

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found that geranic acid is present as a variety-specific compound in Sorachi Ace. However, geranic acid

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could not be detected by GC-O/MS because of its unique characteristics; geranic acid by itself is not

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odor-active, but it functions as an enhancer for other hop-derived flavor compounds. Therefore, the ana-

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lytical strategy for characterizing geranic acid was very different and comprehensive in comparison to

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those used for odor-active unknown compounds (Figure 2). In this work, we report the identification and

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characterization of geranic acid in detail.

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Materials and Methods

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Hop Raw Materials

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The Sorachi Ace and Cascade hops used in this study were harvested in 2014 (Type 90 pellet); the Citra

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and Mosaic hops were harvested in 2015 (Type 90 pellet) and HBC366 (Equanot) was harvested in 2012

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(hop powder) in the U.S. Comet, Hallertau Blanc, and Polaris were harvested in 2014 (Type 90 pellet)

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and Hallertau Tradition (HHT) was harvested in 2015 in Germany (Type 90 pellet). Saaz was harvested

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in 2015 (Type 90 pellet) and Kazbek was harvested in 2014 in the Czech Republic (Type 90 pellet). The

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Aramis, Berbe Rouge, and Triskel hops were bred and grown in 2013 in France (Type 90 pellet). Nelson

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Sauvin was harvested and pelletized in 2015 in New Zealand (Type 90 pellet).

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Pilot-scale Brewing

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Test-brewed beers for analysis of specific compounds in the Sorachi Ace hop. Test-brewed beer A, B, C,

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and D were produced according to the standard method prescribed by the Production & Technology In-

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novation Department, SAPPORO BREWERIES LTD. The wort was prepared using commercially

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available malts and hops in a 100-L pilot scale apparatus. The boiling period was 90 min. To prevent

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over boiling, the HHT hop was added at the beginning of the boiling process (0.2 g of hop/L). For hop

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flavoring or the so-called late-hopping, each hop was added 5 min before the end of the boiling period

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(2.0 g of hop/L cooled wort). Beer A was flavored with Sorachi Ace, beer B with HHT, beer C with

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Saaz, and beer D with Cascade. Each cooled wort was transferred to a fermentation tank (100 L/tank)

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and fermentation was initiated by adding 15.0 × 106 cells/mL of lager yeast (brewery collected; Saccha-

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romyces pastorianus) to the wort. The fermentation temperature was maintained at 10–12 °C (primary

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fermentation). After transferring the fermented wort to another storage tank under a CO2 atmosphere,

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maturation was allowed to occur at 12 °C for 6 days and at 0 °C for 2–3 weeks. Filtration and bottling

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were carried out using the pilot-scale equipment under anti-oxidative conditions. In the case of beer E,

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wort preparation was carried out under the same conditions, except for hop addition. The Sorachi Ace

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hop (0.09 g of hop/L) was added at the beginning of the boiling period. Instead of late-hopping, the

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Sorachi Ace hop (1.5 g of hop/L) was added to the cooled wort together with yeast (dry-hopping). All

99

the hop-flavoring conditions are listed in Table 1.

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Test-brewed beers flavored with various flavor hop varieties. Other test-brewed beers (F to K) were

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produced according to the same method, except that their hop-flavoring conditions were different. For

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late-hopping, each hop was added 5 min prior to the end of the boiling period (0.8 g of hop/L cooled

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wort). Each hop-flavoring condition is described in Table 1. These beers were produced to evaluate the

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brewing performance and flavor characteristics of each variety. In this study, these samples were used

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for comparing the concentrations of the identified compounds in Sorachi Ace and other hop varieties.

106 107

Commercial Beers

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Commercial-scale late-hopped beers (beers L, M, and N) and kettle-hopped beer were brewed by SAP-

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PORO BREWERIES LTD. Beer L was flavored with Mosaic, beer M with Citra and Polaris, and beer N

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with Nelson Sauvin and Hallertau Blanc. The hop-flavoring conditions of these late-hopped beers are

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shown in Table 1. Kettle-hopped pilsner-type beer was used for sensory evaluation as described in the

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following sections (triangular test and profile sensory test).

113 114

Chemicals

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Reference compounds. Geranic acid (>85%, racemic mixture), myrcene (>90%), and (S)-(-)-perillyl al-

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cohol (>96%) were purchased from Sigma Aldrich Japan Co., Ltd. (Tokyo, Japan). Linalool (>96%), α-

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terpinene (>90%), -terpinene (>95%), α-terpineol (>95%, racemic mixture), β-citronellol (>92%, race-

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mic mixture), geraniol (>97%), nerol (>98%), citral (>98%, racemic mixture), (+)-limonene (>95%),

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terpinolene (>85%), farnesol (>95%, mixture of isomers), and benzyl acetate (>99%) were purchased

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from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Methyl geranate (>94%, mixture of isomers)

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was purchased from Thermo Fisher Scientific Inc. (NYSE: TMO).

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Miscellaneous chemicals. Dichloromethane, sodium chloride, sodium hydrogen carbonate, anhydrous

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sodium sulfate, and phosphoric acid were purchased from FUJIFILM Wako Pure Chemical Corporation

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(Osaka, Japan).

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Determination of the Ratio of Isomers in the Reference Compounds by Gas Chromatography-

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Flame Ionization Detection (GC-FID)

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Among the reference compounds, citral, geranic acid, and methyl geranate contained isomers. It is well-

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known that citral is a mixture of geranial and neral. Commercial geranic acid and methyl geranate con-

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tained small amounts of neric acid and methyl nerolate, respectively. All the reference compounds con-

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tained small amounts of contaminants and/or degradation products. For isomer analysis, GC-FID meas-

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urements were conducted on a 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA). The

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carrier gas was helium with a flow rate of 1.7 mL/min in the constant-flow mode. The detector used was

133

a flame-ionization detector at 250 °C. Hydrogen gas at a flow rate of 40 mL/min and air at 450 mL/min

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were used for the FID. Aliquots (1 μL) of each chemical (500 mg/L) were injected into a split injector

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(250 °C; split rate, 100:1; purge flow, 168.5 mL/min) at an oven temperature of 50 °C onto a type HP-

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INNOwax capillary column (30 m × 0.25 mm internal diameter (i.d.); 0.25 μm film thickness; Agilent

137

Technologies). For all measurements, the temperature program was as follows: 50 °C for 2.5 min, heat-

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ed at 10 °C/min to 240 °C, and a 5-min isotherm. The ratio of isomers and contaminants was calculated

139

on the basis of the areas of all the peaks obtained using the FID detector. The calculated isomer ratios

140

are listed in Table 2.

141 142

Analysis of Specific Odor-active Compounds using 1D or 2D GC-O/MS

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Extraction of the test-brewed beer samples. Flavor compounds were extracted according to a method

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previously reported by Tokita et al.23, with some modifications. The Sorachi Ace beer (beer A) and the

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HHT beer (beer B) were selected for GC-O/MS analysis. To extract the acidic volatile fraction (AF),

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each sample beer (200 mL) was saturated with 60 g of sodium chloride, mixed with 200 mL of di-

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chloromethane in a separation funnel, and shaken vigorously for 15 min. The organic solvent layer was

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collected and the aqueous layer was extracted again using another 200 mL of dichloromethane. The re-

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sulting solvents were pooled and dried over 5 g of anhydrous sodium sulfate. The solvents were then

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concentrated to 30 mL using a Vigreux column (60 cm × 1 cm i.d.) at 60 °C. The volatiles were isolated

151

using a solvent-assisted flavor evaporation (SAFE) unit as described by Engel et al24. The distillate was

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concentrated to 1 mL or 100 μL under a stream of nitrogen. To extract the neutral/basic volatile fraction

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(NBF), each sample (200 mL) was neutralized with 4.2 g of sodium hydrogen carbonate and NBF was

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prepared using the same method as described earlier.

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Selectable 1D or 2D GC-O/MS analysis. To analyze the AF and NBF samples, 1D or 2D GC-O/MS was

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performed using a dual low thermal mass gas chromatograph; an olfactory detector port (ODP) sniffing

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port (Gerstel, Mühlheim/Ruhr, Germany) was installed on the 7890A gas chromatograph equipped with

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a 5975C mass spectrometer (Agilent Technologies, Palo Alto, CA). The gas chromatograph was

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equipped with a capillary flow technology (CFT) Deans Switch and a 3-way splitter with a make-up gas

160

line, which were controlled by a pressure-control module (PCM) (Agilent Technologies). The conditions

161

of 1D or 2D GC-O/MS operation were sourced from the protocol described by Tokita et al.22. Separa-

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tions were performed on a DB-WAX (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent Technolo-

163

gies) as the 1D column and a DB-5 (10 m × 0.18 mm i.d., 0.40 μm film thickness; Agilent Technologies)

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as the 2D column. 1D GC-O/MS was used for characterizing the unknown odor-active compounds in

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Sorachi Ace beer by one well-trained panelist. 2D GC-O/MS was used for confirming the odor charac-

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teristics of geranic acid with 3 well-trained panelists.

167 168

Analysis of Specific Compounds by HS-SPME-GC-MS

169

HS-SPME sampling. A Combi-PAL autosampler (CTC Analytics, Zwingen, Switzerland) with an SPME

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fiber (polydimethylsiloxane/divinylbenzene (PDMS/DVB), 65 μm film thickness, Supelco, Bellefonte,

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PA) was used for the extraction of flavor compounds from the beer samples. Firstly, the fiber was pre-

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conditioned at 270 °C for 30 min in the GC injector. Eight milliliters of each test-brewed beer sample

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were transferred into a 20-mL glass vial with 3 g of sodium chloride and the vial was sealed using a

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magnetic cap. After the vial was preincubated while stirring at 40 °C for 15 min, the SPME fiber was

175

inserted into the head space of the vial and held for 15 min for adsorption. After adsorption, the SPME

176

fiber was immediately inserted into a GC injector for thermal desorption for 3 min at 270 °C. Before the

177

next sampling process, the SPME fiber was reconditioned for 15 min at 270 °C in the conditioning sta-

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tion on the Combi-PAL autosampler.

179

GC-MS analysis. GC-MS analysis in the electron impact (EI) mode was performed on a 7890N GC with

180

a 5973 MS (Agilent Technologies). Separation was carried out on a fused silica capillary column DB-

181

WAX (60 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent Technologies). The temperature setting for

182

all the measurements was as follows: 40 °C for 3 min, raised at 5 °C/min to 250 °C, and a 5-min iso-

183

therm. The injection port, equipped with a 0.75 mm i.d. liner (Supelco), was maintained at 270 °C. The

184

inlet was operated in the splitless mode and the injection purge on the GC was off initially for 3 min for

185

injecting the SPME fiber. The flow rate of the helium carrier gas was 2.1 mL/min in the constant-flow

186

mode. The mass spectrometer was operated under the following conditions – ionization voltage of 70 eV

187

(EI), ion source temperature of 230 °C, quadrupole temperature of 150 °C, and transfer line temperature

188

of 250 °C. An MS detector in the full-scan mode was used for acquiring the data. Electron ionization

189

mass spectrometric data was collected in the m/z range of 29 to 280.

190 191

Identification of Hop-derived Compounds

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All the hop-derived compounds were identified by comparing their retention indices (RIs), odor qualities,

193

and mass spectra (MSs) with those of authentic chemical standards available in the laboratory using the

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same instrument. RIs were calculated after analyzing C6-C24 n-alkane series (Supelco) under the same

195

chromatographic conditions. Before identification by comparison with MSs of authentic chemical stand-

196

ards, each targeted substance was estimated using the NIST 05.L Database (Agilent Technologies).

197 198

Quantitation of Hop-Derived Compounds by HS-SPME-GC-MS

199

Quantitation of terpene alcohols, aldehydes, esters, and hydrocarbons. The quantitation of terpene alco-

200

hols, aldehydes, esters, and hydrocarbons was conducted on a 6890N GC with a 5973N MS (Agilent

201

Technologies). A SPME fiber (PDMS/DVB, 65 μm film thickness, Supelco) was used for volatile-

202

compound extraction. Eight milliliters of each beer sample were placed in a 20-mL glass vial with 3 g of

203

sodium chloride, followed by spiking with 40 L of 10 mg/L benzyl acetate as the internal standard. The

204

vial was hermetically sealed with a magnetic cap and agitated at 40 °C for 15 min on a Combi-PAL au-

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tosampler (CTC Analytics). The SPME fiber was inserted into the head space of the vial and held for 15

206

min for adsorption. After adsorption, the SPME fiber was immediately inserted into a GC injector for

207

thermal desorption for 3 min at 270 °C. The volatiles were injected in the splitless mode. The separation

208

of volatiles was performed on a DB-WAX column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Ag-

209

ilent Technologies) with a helium carrier gas at a constant flow rate of 1.2 mL/min. The oven tempera-

210

ture setting was as follows: 40 °C for 3 min, raised to 250 °C at a rate of 5 °C/min, and a 5-min isotherm.

211

The mass spectrometer functioned in the EI mode (70 eV) and was operated in the selected ion monitor-

212

ing (SIM) mode. Calibration curves were constructed using the beer samples spiked with reference

213

compounds. An appropriate range of the calibration curves was determined for calculating the concen-

214

trations of the compounds in sample beers. The monitored ions, range of the calibration curve, and the

215

ratios of the isomers in the reference compounds are listed in Table 2. All calibrations produced a linear

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correlation with an R2 value >0.99, across the entire concentration range analyzed. All tests were run

217

twice to reduce error.

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Quantitation of geranic acid and neric acid. Geranic acid and neric acid were quantitated on a 6890N

219

GC with a 5973N MS (Agilent Technologies). Four milliliters of each test-brewed beer sample and 4

220

mL of 0.1% (v/v) aqueous phosphoric acid were added to a 20-mL glass containing 3 g of sodium chlo-

221

ride, followed by spiking with 20 L of 10 mg/L benzyl acetate as the internal standard. The vial was

222

hermetically sealed using a magnetic cap and agitated at 60 °C for 15 min on a Combi-PAL autosampler

223

(CTC Analytics). The SPME fiber (PDMS/DVB, 65 μm film thickness, Supelco) was inserted into the

224

head space of the vial and held for 15 min for adsorption. After adsorption, the SPME fiber was imme-

225

diately inserted into a GC injector for thermal desorption for 3 min at 270 °C. The volatiles were inject-

226

ed in the splitless mode. The separation of volatiles was performed on a DB-FFAP (30 m × 0.25 mm i.d.,

227

0.25 μm thickness; Agilent Technologies) with a helium carrier gas at a constant flow rate of 1.0

228

mL/min. The oven temperature was increased from 40 °C (held for 3 min) to 250 °C at a rate of

229

5 °C/min. The mass spectrometer functioned in the EI mode (70 eV) and was operated in the SIM mode.

230

Calibration curves were constructed using test-brewed beers containing standard substances at concen-

231

trations of 10, 25, 50, 100, 250, 500 and 1000 μg/L. The monitored ions, range of the calibration curves,

232

and ratios of the isomers are listed in Table 2. All calibrations indicated a linear correlation with an R2

233

value of >0.99 over the entire concentration range analyzed. All tests were run twice to reduce error.

234 235

Sensory Evaluation by Triangular Test

236

The model beers were prepared by spiking a commercial pilsner-type beer made with kettle-hopping

237

with the flavor compounds. The concentrations of the spiked compounds were adjusted to 3 times of the

238

values obtained from the test-brewed beer late-hopped with Sorachi Ace. Two different sets of model

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beers were prepared. In the set of model 1, the control sample was an unspiked pilsner-type beer and the

240

test sample was a pilsner beer spiked with only geranic acid (including neric acid); in the set of model 2,

241

the control sample was a beer spiked with terpene alcohols (linalool and geraniol) and the test sample

242

was a beer spiked with both geranic acid and terpene alcohols. Sensory triangular tests were carried out

243

using these model sets by 9 well-trained panelists. The room was air-conditioned at 23 °C. The samples

244

were stored at 4 °C and 70 mL of each sample was poured in uncovered glass vessels (total volume =

245

150 mL) immediately before presenting them to the panelists. The significance of the results was deter-

246

mined according to the binomial law.

247 248

Determination of Olfactory Threshold

249

The sensory evaluation was performed by 15 well-trained panelists. Perception threshold of geranic acid

250

(involving neric acid) was assessed by a forced-choice ascending concentration series method of limits25.

251

Briefly, the directional triangular tests of six increasing concentrations in model carbonated solution

252

(0.1 %v/v ethanol, 3.0 kg/cm2 pressure). Then 50 mL of each sample solution was presented in plastic

253

cups. The best estimate threshold was calculated for each panelist as the geometric mean of the highest

254

concentration missed and the next highest concentration. The group threshold was calculated as the ge-

255

ometric mean of the best estimate thresholds of the panelists.

256 257

Profile Sensory Test

258

In order to analyze the synergy between geranic acid and hop-derived terpenoids, profile sensory tests

259

were carried out on model beers; the procedure is as follows (Table 6). Japanese commercial pilsner-

260

type beer made with kettle-hopping was used as the control beer. The concentrations of the targeted

261

compounds in the simulated model beers were similar to their concentrations in the dry-hopped Sorachi

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Ace beer (beer E). The concentrations of not only the major compounds but also the minor compounds

263

were calculated at trace levels from the calibration curves in SPME-GC-MS, in order to simulate the

264

Sorachi Ace beer as closely as possible. The model beer GA was prepared by spiking the control beer

265

with a mixture of carboxylic acids. The model beer T contained hop-derived terpenoids, including a

266

mixture of alcohols, aldehydes, esters, and hydrocarbons. The model beer T+GA contained all mixtures;

267

a mixture of carboxylic acids, alcohols, aldehydes, esters, and hydrocarbons. These samples were stored

268

at 4 °C and 70 mL of each sample was poured in uncovered glass vessels (total volume = 150 mL) im-

269

mediately before presenting them to the panelists. Later, the six flavor characters (flowery, fruity, lemon,

270

tropical, green, and woody) of each sample were scored from 0 (no flavor) to 3 (strong flavor) at inter-

271

vals of 0.5. The mean intensity value of each characteristic was calculated and paired t-tests were con-

272

ducted on Microsoft Excel 2013.

273 274

Results and Discussion

275

Analysis of Variety-Specific Compounds in Test-Brewed Beer made with the Sorachi Ace Hop

276

Investigation of odor-active compounds by 1D GC-O/MS. To evaluate the variety-specific compounds

277

derived from the Sorachi Ace hop, we brewed 5 beers (beer A to E, Table 1). In the late-hopped test-

278

beers, the Sorachi Ace beer (beer A) could be clearly distinguished from the HHT beer (beer B), Saaz

279

beer (beer C), and Cascade beer (beer D), owing to its characteristic “lemongrass-like” aroma. The aro-

280

ma of dry-hopped Sorachi Ace beer (beer E) was more prominent than that of the late-hopped beer (beer

281

A). However, we selected beer A to investigate the variety-specific compounds in Sorachi Ace beer.

282

This is because our panelists could recognize the varietal aroma of the Sorachi Ace beer, both dry-

283

hopped and late-hopped; the dry-hopped beer contained large amounts of various hop-derived com-

284

pounds, which could prevent the separation/purification of the targeted compounds. In our previous

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studies9-12, we demonstrated that characteristic key compounds could be relatively easily discovered us-

286

ing the less-characteristic variety as a reference sample. Therefore, we selected beer B as the control

287

beer.

288

The Sorachi Ace beer (beer A) and the HHT beer (beer B) were analyzed by 1D GC-O/MS. Later, we

289

selected several zones whose odor was strong in only the Sorachi Ace sample, but not in the HHT sam-

290

ple; the same consideration was applied with respect to flavor. The odor-active compounds, which were

291

detected in these zones, were identified by a library search of the mass spectra using the NIST 05.L Da-

292

tabase and by comparing the RIs, MSs, and sensory attributes with the reference compounds (Table 3).

293

We expected that the test-brewed Sorachi Ace beer would include several monoterpene alcohols, such as

294

linalool, geraniol, and β-citronellol, at higher quantities than the HHT beer. It is known that certain ter-

295

pene alcohols have a floral and/or citrus-like characteristic flavor. Takoi et al. reported that the lime-like

296

citrus aroma of beer could be attributed to the presence of coexisting linalool, geraniol, and -

297

citronellol13. Among the identified compounds in Table 3, these terpene alcohols were detected in the

298

Sorachi Ace beer. It was assumed that these compounds could contributed to the variety-specific flavor

299

characteristics of the Sorachi Ace beer, for example, its citrus and/or lemongrass-like characteristics.

300

However, the flavor characteristics of other compounds (Table 3) were different from those typical of

301

the Sorachi Ace beer, especially the pine-like and/or woody characteristics. Therefore, we speculated

302

that the specific flavor of the Sorachi Ace beer could not be completely explained by the identified com-

303

pounds described in Table 3.

304

In general, GC-O is suitable for identifying unknown compounds at a low threshold, for example vola-

305

tile thiols9-11. Although it has been reported that relatively large amounts of 4-methyl-4-sulfanylpentan-

306

2-one (4MSP) are included in the Sorachi Ace hop20, we could not identify a corresponding zone by 1D

307

GC-O/MS. 4MSP, a volatile thiol with a very low threshold (1.2 ng/L16), is often found in flavor hops10,

14


Page 15 of 38


308

11, 20, 21, 26

309

shown); previous studies also made a similar observation21. In addition, the characteristic aroma of the

310

Sorachi Ace beer was different from that of 4MSP, whose flavor is somewhat reminiscent of blackcur-

311

rant and/or muscat26. Any zones corresponding to other volatile thiols, for example 3-sulfanylhexan-1-

312

ol10, 11, 20 and/or 3-sulfanyl-4-methylpentan-1-ol10, 11, were not recognized by 1D GC-O/MS. Therefore,

313

we assumed that the unknown compounds in the Sorachi Ace beer might be overlapping with other

314

compounds having a strong odor on this DB-WAX column (30 m × 0.25 mm i.d., 0.25 μm film thick-

315

ness); further, the GC-O technique might not be capable of detecting relatively high thresholds.

316

Identification of geranic acid using HS-SPME-GC-MS. As the results of GC-O analysis were not suffi-

317

cient to identify the flavor compounds specific only to the Sorachi Ace beer, we tried to evaluate these

318

compounds using another approach. To this end, we performed HS-SPME-GC-MS analysis using a 60

319

m DB-WAX column. Similar to 1D GC-O/MS analysis, the Sorachi Ace beer (beer A) and HHT beer

320

(beer B) were analyzed to compare their chromatograms. From the HS-SPME-GC-MS analysis results

321

on the 60 m DB-WAX column, we found “geranic acid” (IUPAC; 3,7-dimethyl-2,6-octadienoic acid,

322

CAS No. 459-80-3) only in the Sorachi Ace beer, but not in the HHT beer. The chromatograms obtained

323

on the 30 m and 60 m DB-WAX columns are shown in Figure 3. Geranic acid could be separated from

324

9-decenoic acid using the 60 m DB-WAX column (Figure 3-II), while their peaks overlapped when the

325

30 m DB-WAX column was used (Figure 3-I). The RI of geranic acid in beer A was determined to be

326

2349. The MSs of the obtained peaks and the reference compounds (geranic acid) are shown in Figure 4.

327

However, we could not detect the odor of geranic acid using 1D GC-O/MS on the 30 m DB-WAX col-

328

umn, because its peak overlapped with that of 9-decenoic acid, whose odor was stronger. Therefore, we

329

tried to confirm whether the odor of geranic acid could be detected by 2D GC-O/MS using same extract-

330

ed sample of the Sorachi Ace beer used for 1D GC-O/MS analysis and as the reference compound. The

. The Sorachi Ace hop used in this study contained a very small amount of 4MSP (data not

15



Page 16 of 38

331

peak corresponding to geranic acid could be separated from that of 9-decenoic acid with heart-cutting of

332

the overlapped peak in 2D GC-O/MS. However, well-trained panelists could not detect the odor of gera-

333

nic acid in 2D GC-O/MS.

334

It has been proposed that geranic acid is a derivative of geraniol and geranial, which are known as hop-

335

derived flavor compounds, especially present in flavor hops7, 11-17, 19, 20. In previous studies27-34, geranic

336

acid was found in lemongrass, ginger, lemon oil, tomato, and grape, which are raw materials used for

337

wine making. Methyl geranate, which is a methyl ester of geranic acid, has been reported as a compound

338

derived from hop oil19, 20, 35, 36. Roberts et al. assumed geranic acid to be a possible precursor of methyl

339

geranate36, which they first identified in hop oil35. Peacock et al. analyzed three commercial beers and

340

detected geranic acid in only one of the beer samples at trace levels (1 μg/L)37. Our study is the first to

341

report the presence of hop-derived geranic acid in Sorachi Ace beers at a significant level.

342 343

Quantification of Hop-Derived Key Compounds using HS-SPME-GC-MS in Test-brewed Beers

344

As described earlier, geranic acid is regarded as a derivative of geraniol and geranial. It is thought that

345

the biosynthesis of geraniol derivatives in a hop cone occurs as follows. Geraniol is firstly oxidized to

346

geranial, which is subsequently oxidized to geranic acid, and finally geranic acid is esterified to methyl

347

geranate (Figure 5). It has been reported that the sedge Cyperus iria followed the biosynthesis pathway

348

from farnesol to methyl farnesoate30. The structure of farnesol is similar to that of geraniol; therefore,

349

not only farnesol but also geraniol could follow this reaction pathway. For example, Gewürztraminer

350

grapes contain methyl farnesoate, methyl geranate, and geranic acid28. It is thought that the hop plant

351

also adopts this biosynthesis pathway, due to the presence of methyl geranate in hops.

352

These geraniol-derivatives contain stereoisomers of nerol, neral, neric acid, and methyl nerolate. In addi-

353

tion, using GC-O/MS, we found that high concentrations of geraniol and linalool might be included in

16


Page 17 of 38


354

the Sorachi Ace beer. Therefore, we selected the following geraniol-derivatives as the target flavor com-

355

ponents – terpene alcohols (geraniol, nerol), terpene aldehydes (geranial and neral), carboxylic acids

356

(geranic acid and neric acid), and esters (methyl geranate and methyl nerolate). In addition, linalool was

357

also selected as a target compound because of its role in hop aroma development12-17. These compounds

358

were analyzed in the sample beers (beer A to N in Table 1), which were made using 13 or more different

359

hops, by HS-SPME-GC-MS. The limits of detection (LODs) and limits of quantitation (LOQs) deter-

360

mined in the beers were 10 μg/L and 25 μg/L for geranic acid and neric acid and 0.3 μg/L and 1 μg/L for

361

other terpenoids, respectively (Table 2). The concentration of each isomer was calculated using the rati-

362

os described in Table 2. The concentrations of these compounds in the 14 sample beers are shown in

363

Table 4.

364

Finally, it could be confirmed that geranic acid is found only in the Sorachi Ace beers (beer A and E) at

365

significant levels (133 μg/L and 178 μg/L, respectively). Although this compound was present only at

366

trace levels in the Mosaic beer (beer L), it was not detected in other sample beers. Thus, it could be con-

367

firmed that geranic acid is very specific to Sorachi Ace beers. Peacock et al. previously reported that 1

368

μg/L of geranic acid was detected in an American commercial beer37. Our investigation is the first to

369

report the occurrence of geranic acid in Sorachi Ace beers at very high levels (133 μg/L and 178 μg/L).

370

The concentration of methyl geranate in the Sorachi Ace beer was at trace levels. In addition, the con-

371

centrations of geraniol and linalool were relatively higher in the Sorachi Ace beers than in other beers.

372

Therefore, we developed a hypothesis that terpene alcohols (geraniol and linalool) along with geranic

373

acid, might be regarded as possible contributors for the varietal aroma of Sorachi Ace in the 9 target fla-

374

vor compounds.

375 376

Additive effect between Geranic Acid and Terpene Alcohols

17



Page 18 of 38

377

In the field of flavor science, it is well known that there is an additive effect between classes of com-

378

pounds with similar structures38, 39. For example, 3-sulfanylhexan-1-ol (C6) can enhance the odor inten-

379

sities of 3-sulfanypentan-1-ol (C5) and 3-sulfanylheptan-1-ol (C7)39. On the other hand, the intensities

380

of several coffee aroma compounds could be enhanced by certain carboxylic acids, such as acetic acid

381

(C2) and butyric acid (C4), at subthreshold levels 40, 41. From these studies38-41, we assumed that geranic

382

acid can enhance the intensities of geraniol-derivatives at subthreshold levels, at which well-trained pan-

383

elists could not recognize the odor of geranic acid itself. Because geranic acid is similar in structure to

384

geraniol-derivatives (Figure 5) and it is also one of the carboxylic acids.

385

In this section, we selected several candidate flavor components as contributors for the varietal aroma of

386

Sorachi Ace; these include terpene alcohols, which were present in the Sorachi Ace beer at relatively

387

high levels (Table 4) and recognized by GC-O analysis (Table 3) and geranic acid, which was identified

388

as a specific compound, although its odor could not be detected by GC-O. We added geranic acid and/or

389

terpene alcohols (linalool and geraniol) to kettle-hopped beer and confirmed that the flavor of the beer

390

containing all these compounds was similar to that of the Sorachi Ace beer, unlike the beers containing

391

only geranic acid or only the terpene alcohols (data not shown). Therefore, a sensory triangular test was

392

conducted to understand the impact of geranic acid on the aroma of beers.

393

We investigated whether geranic acid could contribute to the flavor of beer. Table 5 presents the results

394

of 2 sets of sensory triangular tests. In the set of model 1 (control, kettle-hopped beer; test, kettle-hopped

395

beer with geranic acid), well-trained panelists could not recognize the difference between the control

396

and test samples (no significant difference). However, in the set of model 2 (control, kettle-hopped beer

397

with linalool and geraniol; test, kettle-hopped beer with linalool, geraniol, and geranic acid), 7 of the 9

398

panelists could recognize the test sample containing geranic acid at a significant level with a risk of 1%.

399

Therefore, we surmised that geranic acid itself had little direct effect on the beer flavor, but could prob-

18


Page 19 of 38


400

ably enhance the flavor intensities of linalool and geraniol, despite its own very low flavor intensity. In

401

addition, the geranic acid-spiked test sample in model 2 had citrus (lemon-like) and/or woody notes ac-

402

cording to the well-trained panelists.

403

The olfactory threshold of standard geranic acid (involving neric acid) used in this study was 2.2 mg/L

404

in model carbonated solution by 15 well-trained panelists. The odor intensity of geranic acid itself was

405

quite low and the perceptive aroma was slightly oily, floral, and plant-like, according to the panelists.

406

The concentration of geranic acid 399 μg/L we spiked was less than a third of the olfactory threshold.

407

However, our panelists could recognize the test sample in model 2, despite the concentration of geranic

408

acid being at subthreshold levels. Therefore, it is suggested that geranic acid is characteristic of the

409

Sorachi Ace hop and that this compound is a key contributor to the varietal aroma of Sorachi Ace due to

410

its coexistence with other flavor compounds, such as linalool and geraniol. In order to investigate

411

whether geranic acid can function as an enhancer under emphasized conditions, the concentrations of the

412

spiked compounds were adjusted at threefold amounts to those of same compounds quantitated in the

413

late-hopped Sorachi Ace beer (beer A) in this sensory test. Next, we evaluated the effect of geranic acid

414

on other hop-derived terpenoids using model beers containing spiked compounds at concentrations simi-

415

lar to those in the dry-hopped Sorachi Ace beer (beer E).

416 417

Study of the Synergy between Geranic acid and Hop-Derived Terpenoids

418

We selected the dry-hopped Sorachi Ace beer (beer E) as the target beer to simulate the varietal aroma

419

of Sorachi Ace, because the Sorachi Ace hop is mainly used for dry-hopped craft beers. To simulate

420

beer E, we selected various hop-derived compounds, not only the geraniol-derivatives shown in Table 4,

421

but also terpene hydrocarbons, myrcene, α-terpinene, -terpinene, (+)-limonene, and terpinolene, as

422

these compounds, which exhibit resinous and/or woody odors, can be enriched in beers by dry-hopping;

19



Page 20 of 38

423

further, their structures are similar to those of geraniol-derivatives. In order to evaluate any possible

424

synergy between subthreshold compounds, the concentrations of several compounds contained at trace

425

levels in beer E were calculated using the HS-SPME-GC-MS calibration curves and these compounds

426

were used to spike the model beers. Of these selected compounds, the spiked concentrations of neral,

427

neric acid, and methyl nerolate did not correspond with those calculated for beer E, as we used commer-

428

cially available reference compounds of citral, geranic acid, and methyl geranate, which also contained

429

their isomers. The compositions of the spiked compounds in the model beers are listed in Table 6. The

430

model beers were evaluated by a profile sensory test by 8 well-trained panelists.

431

Figure 6 shows the six flavor characters (flowery, fruity, lemon, tropical, green, and woody) of the con-

432

trol and model beers. All the six characters in the control beer were at very low levels. In the case of the

433

model beer GA, which was spiked with only geranic acid and neric acid, the average scores of “Flow-

434

ery,” “Fruity,” and “Green” slightly increased; however, all the scores were at low levels. In the model

435

beer T, which contained hop-derived terpenoids, except for carboxylic acid, the scores of “Flowery,”

436

“Fruity,” “Lemon,” “Green,” and “Woody” were clearly higher. In the model beer T+GA, which was

437

spiked with all the compounds, the “Flowery,” “Fruity,” “Lemon,” “Tropical,” and “Green” characters

438

were at higher scores than those in the model beer T. Especially, the scores of “Flowery” and “Lemon”

439

were significantly higher with a risk of 5 %, when compared to the model beer T (Table 7). This result

440

suggested that geranic acid can enhance the intensities of other hop-derived terpenoids and also change

441

the flavor characters of beers. In addition, the characters of the model beer T+GA were close to those of

442

the test-brewed beer E. The characters of beer E could be successfully reconstructed by spiking with se-

443

lected hop-derived terpenoids, including geranic acid. On the basis of these results, we propose that ge-

444

ranic acid is a key compound responsible for the varietal aroma of the Sorachi Ace hop. Finally, it was

20


Page 21 of 38


445

concluded that geranic acid exhibits very unique characteristics; by itself, it was less odor-active, but

446

functioned as an enhancer for hop-derived terpenoids at subthreshold levels.

447 448

Abbreviations

449

AF, acidic volatile fraction; CFT, capillary flow technology; 1D or 2D GC-O/MS, one-dimensional or

450

two-dimensional gas chromatography-olfactometry/mass spectrometry; EI, electron impact; HHT, Hal-

451

lertau Tradition; HS-SPME-GC-MS, head space-solid phase microextraction-gas chromatography-mass

452

spectrometry; LOD, limit of detection; LOQ, limit of quantitation; NBF, neutral/basic volatile fraction;

453

ODP, olfactory detector port; PCM, pressure control module; PDMS/DVB, polydimethylsilox-

454

ane/divinylbenzene; RIs, retention indices; SAFE, solvent-assisted flavor evaporation; SIM, selected ion

455

monitoring

456 457

Acknowledgements

458

We thank Yakima Chief Hopunion, LLC, John I. Haas, Inc., Hop Breeding Company, LLC, S. S. Steiner,

459

Inc., Joh. Barth & Sohn GmbH & Co. KG, Bohemia Hop a.s., Comptoir Agricole, and New Zealand

460

Hops Ltd. for supplying hop samples. We gratefully acknowledge all the panelists at SAPPORO

461

BREWERIES LTD. and SAPPORO HOLDINGS LTD. for their sensory work. The kind help provided

462

by Yutaka Itoga at the Bioresources Research & Development Department is also acknowledged.

463 464

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465

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573

9901.

574

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Page 27 of 38


Figure Captions Brewer’s Gold (♀) X Saaz (♂) ↓ 　　　70K-SH6 (♀) X Beikei 2 (♂) ↓ SORACHI ACE

Figure 1. Crossing history of Sorachi Ace hop.

27



Sorachi Ace hop/beer unknown specific compounds odor-active compound? overlapped? less odor-active?

GC-O/MS DB-WAX (30 m)

SPME-GC-MS DB-WAX (60 m)

selected compound linalool, geraniol etc. odor-active not specific for Sorachi Ace

identification of

'geranic acid' 'geranic acid' specific for Sorachi Ace not detected in GC/O! less odor-active

quantification of selected compound

quantification of 'geranic acid'

sensory triangular test

'geranic acid' as an enhancer

profile sensory test

'geranic acid' as a key compound for varietal aroma of Sorachi Ace

Figure 2. Identification and characterization strategy in this study.

28


Page 28 of 38

Page 29 of 38


Beer A (Sorachi Ace)

I. 30 m

Beer B (HHT)

coelution of geranic acid and 9-decenoic acid

9-decenoic acid

9-decenoic acid 9-decenoic acid II. 60 m

geranic acid

Figure 3. Comparison of GC-MS peaks obtained from the analyses of the test-brewed beers: ‘I. 30 m’, GC-MS charts on a DB-WAX (30 m × 0.25 mm i.d., 0.25 μm film thickness); ‘II. 60 m’, GC-MS charts on a DB-WAX (60 m × 0.25 mm i.d., 0.25 μm film thickness); Beer A, late-hopped with Sorachi Ace; Beer B, late-hopped with HHT.

29



A

Page 30 of 38

30m DB-WAX

B

60m DB-WAX

C O OH

geranic acid RI 2349

Figure 4. Mass spectra of the peak overlapped with 9-deconoic acid and geranic acid on 30 m DA-WAX (A), the separated peak of geranic acid on 60 m DB-WAX (B) and reference compound of geranic acid (C).

30


Page 31 of 38


OH geraniol

O geranial

O OH geranic acid

O O

methyl geranate Figure 5. Possible biosynthesis pathway of geraniol, geranial, geranic acid, and methyl geranate in plants.

31



Flowery

Flowery

2.0

Woody

1.5

1.0

Flowery

2.0

Fruity

Woody

1.5

2.0

Fruity

1.0

Woody

1.5

1.0

0.5

0.5

0.5

0.0

0.0

0.0

Green

Lemon

Green

Lemon

Green

Tropical

Tropical

control beer

model beer GA

model beer T

Flowery

Flowery

1.5 1.0

Flowery

2.0

Fruity

Woody

1.5

2.0

Fruity

1.0

Woody

1.5 1.0

0.5

0.5

0.5

0.0

0.0

0.0

Green

Lemon

Green

Fruity

Lemon

Tropical

2.0

Woody

Page 32 of 38

Lemon

Green

Fruity

Lemon

Tropical

Tropical

Tropical

model beer T+GA (vs model beer T)

model beer T+GA (vs beer E)

beer E

Figure 6. Flavor profile of model beers containing mixture of hop-derived flavor compounds and/or geranic acid: control beer, Japanese commercial kettle-hopped beer; model beer GA, spiking with geranic acid; model beer T, spiking with selected hop-derived flavor compounds; model beer T+GA, spiking with all compounds; dotted line, scores in model beer T (model beer T+GA (vs T)) or test-brewed beer E dry-hopped with Sorachi Ace (model beer T+GA (vs beer E)).

32


Page 33 of 38


Tables Table 1. Hop-flavoring conditions in test-brewed beers and commercial beers.

flavoring hop

beer

hop variety

hop type

origin

grown area

form

hopping

hop dosage (g of hop/L)

A B C D

Sorachi Ace HHT Saaz Cascade

flavor hop aroma hop aroma hop flavor hop

Japan Germany Czech U.S.

U.S. Germany Czech U.S.

T90 T90 T90 T90

late late late late

2.0 2.0 2.0 2.0

E

Sorachi Ace

flavor hop

Japan

U.S.

T90

dry

1.5

F G H I J K

Aramis Berbe Rouge Comet HBC366 (Equanot) Kazbek Triskel

flavor hop flavor hop flavor hop flavor hop flavor hop flavor hop

France France U.S. U.S. Czech France

France France Germany U.S. Czech France

T90 T90 T90 powder T90 T90

late late late late late late

0.8 0.8 0.8 0.8 0.8 0.8

L*

Mosaic

flavor hop

U.S.

U.S.

T90

late

1.1

M*

Citra Polaris

flavor hop flavor hop

U.S. Germany

U.S. Germany

T90 T90

late late

0.8 0.3

N*

Nelson Sauvin Hallertau Blanc

T90 T90

late late

1.1 0.1

flavor hop New Zealand New Zealand flavor hop Germany Germany

*Japanese commercial beer; HHT, Hallertauer Tradition

33



Page 34 of 38

Table 2. Gas chromatography-mass spectrometry parameters for quantitation of selected flavor compounds.

group

compound

isomers

ratio of quantifier ion

qualifier ion

isomers

(m/z )

(m/z )

column

R2

slope

linear range

(μg/L)

LOD

LOQ

(μg/L) (μg/L)

linalool α-terpineol β-citronellol

-

-

136 136 138

121 121 109

DB-WAX DB-WAX DB-WAX

1.000 1.000 1.000

867 823 2964

1-100 1-100 1-100

0.3 0.3 0.3

1 1 1

geraniol nerol

-

-

136 121

121 93, 69

DB-WAX DB-WAX

1.000 1.000

4088 3650

1-100 1-100

0.3 0.3

1 1

aldehydes

citral

geranial neral

0.53 0.27

152 137

137 119

DB-WAX DB-WAX

0.999 1.000

6535 16794

0.5-50 0.3-30

0.3 0.3

1 1

carboxylic acids

geranic acid

geranic acid neric acid

0.7 0.2

123 123

168, 100 168, 100

DB-FFAP DB-FFAP

1.000 0.999

7082 8636

7-700 10-200

10 10

25 25

esters

methyl geranate

methyl geranate methyl nerolate

0.715 0.285

114 114

182 182

DB-WAX DB-WAX

1.000 1.000

2335 243

1-70 0.3-30

0.3 0.3

1 1

hydrocarbons

α-terpinene γ-terpinene (+)-limonene terpinolene myrcene

-

-

136 136 136 136 93

121 121 121 121 69

DB-WAX DB-WAX DB-WAX DB-WAX DB-WAX

0.996 0.996 0.995 0.997 0.996

118 91 265 56 67

1-5 1-5 1-5 1-5 1-100

0.1 0.1 0.1 0.1 0.3

0.3 0.3 0.3 0.3 1

-

benzyl acetate (ISTD)

-

-

108

91

-

-

-

-

-

-

alcohols

LOD, limit of detection;　LOQ, limit of quantititation

34


Page 35 of 38


Table 3. Retention indices and odor qualities of identified flavor compounds by GC-O analysis on 1D DB-WAX column.

odor qualitiesb

DB-WAXa

compound

Sorachi Ace Beer (beer A)

HHT beer (beer B)

IDc

1553 1774 1856 2016 2338

linalool β-citronellol geraniol (S )-(-)-perillyl alcohol farnesol

floral, acrid-smelling citrus floral, flowery sweet, floral floral, powdery

floral, acrid-smelling (weak) floral, flowery (none) (none)

MS, RI, CI MS, RI, CI MS, RI, CI MS, RI, CI MS, RI, CI

HHT, Hallertauer Tradition a

Retention Index on a DB-WAX column

b

Odor Qualities percieved at the olfactory port

c

Identification Method: MS, mass spectral identification; RI, retention index confirmation with olfactory event; CI, chemical standard identification using pure analytical standards

Table 4. Concentrations of selected flavor compounds in test-brewed beers made using various hops. alcohols

aldehydes

carboxylic acids

linalool

geraniol

nerol

geranial

neral

esters

beer

flavoring hop

hopping

(μg/L)

(μg/L)

(μg/L)

(μg/L)

(μg/L)

geranic acid (μg/L)

A B C D

Sorachi Ace HHT Saaz Cascade

late late late late

70 60 16 42

16 5.2 5.5 43

4.1 1.4 1.2 3.1

tr tr tr tr

n.d. n.d. n.d. n.d.

133 n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

tr tr tr 3.1

n.d. n.d. n.d. n.d.

E

Sorachi Ace

dry

97

98

9.6

2.7

tr

178

n.d.

1.2

n.d.

F G H I J K

Aramis Berbe Rouge Comet HBC366 (Equanot) Kazbek Triskel

late late late late late late

42 100 32 25 11 52

5.7 11 17 15 5.3 7.7

1.3 2.2 1.4 2.6 0.8 1.9

n.d. tr tr tr tr tr

n.d. n.d. n.d. n.d. n.d. n.d.



3.9 17 2.4 7.7 tr 10

n.d. tr n.d. n.d. n.d. n.d.

L M N

Mosaic Citra* Nelson Sauvin*

late late late

74 87 43

21 25 9.1

4.8 4.4 1.7

2.8 2.3 1.2

n.d. n.d. n.d.

tr n.d. n.d.

n.d. n.d. n.d.

51 32 2.2

1.6 tr n.d.

*flavoring together with other hop (see Table 1); HHT, Hallertauer Tradition; tr, trace; n.d., not detected

35


neric acid (μg/L)

methyl geranate (μg/L)

methyl nerolate (μg/L)


Page 36 of 38

Table 5. Triangular test involving 9 panels (in model sample based on kettle-hopped commercial beer).

Sample

test

Model 1

beer + 399 μg/L geranic acid

Model 2

beer + 210 μg/L linalool + 49 μg/L geraniol + 399 μg/L geranic acid

control

correct answers/ total answers

p

free comments of correct answers

beer

2/9

-

-

7/9

0.01

green woody lemon-like

beer + 210 μg/L linalool + 49 μg/L geraniol

Table 6. Concentrations of spiked compounds in model beers for profile sensory test. concentraions of spiked compounds

alcohols

aldehydes

carboxylic acids

hydrocarbons

concentrations in dry-hopped

ratioa (%)

Sorachi Ace beer (test-brewed beer E)

control beer

model beer GA

model beer T

model beer T+GA

linalool α-terpineol β-citronellol

(μg/L) (μg/L) (μg/L)

-

97 20 13

-

-

97 20 13

97 20 13

geraniol nerol

(μg/L) (μg/L)

-

98 9.6

-

-

98 9.6

98 9.6

geranial

(μg/L)

53

2.7

-

-

2.7

c

2.7

c

1.4

1.4

-

178c 51c

b

neral

(μg/L)

27

tr (0.46)

-

-

geranic acid

(μg/L)

70

178

-

178c

neric acid

esters

isomer

(μg/L)

methyl geranate (μg/L) methyl nerolate (μg/L)

20

n.d.

71.5

1.2

c

c c

-

51

-

-

-

1.2c

1.2c

c

0.5c

28.5

b

tr (0.01)

-

-

0.5

α-terpinene

(μg/L)

-

tr (0.20)b

-

-

0.2

0.2

γ-terpinene (+)-limonene

(μg/L) (μg/L)

-

tr (0.03)b 0.26

-

-

0.03 0.26

0.03 0.26

terpinolene myrcene

(μg/L) (μg/L)

-

tr (0.06) 10

b

-

-

0.06 10

0.06 10

tr, trace; n.d., not detected a

isomer ratio in standard substances (geranial, geranic acid, and methyl geranate), omitting degradation products and/or contaminants.

b

concentrations of 'tr' compounds were calculated according to the caｌibration curves in SPME-GC-MS for profile sensory test.

c

concentrations of spiked standard substances (geranial, geranic acid, and methyl geranate) were adjusted according to the concentrations of major isomers.

36


Page 37 of 38


Table 7. Intensity and t-test value for profile sensory test of model bees.

a

intensity

flowery fruity lemon tropical green woody a

p

b

control beer

test-brewed beer E

model beer GA

model beer T

model beer T+GA

model beer T:T+GA

0.38 0.38 0.00 0.00 0.38 0.00

1.50 1.50 1.50 1.13 1.38 1.00

1.00 0.63 0.25 0.25 0.50 0.50

1.25 1.25 0.75 0.13 0.63 0.88

1.88 1.50 1.75 0.50 1.00 0.88

0.049 0.351 0.018 0.080 0.080 1.000

mean intensity value of the scores from eight well-trained panels

b

paired t -test comparing model beer T and T+GA. *, significant difference with a risk of 5 %

37


* *


Table of Contents Graphics (TOC)

38


Page 38 of 38

Identification and Characterization of Geranic acid as Unique Flavor

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