Determination of Linalool in Different Hop Varieties Using a New Method Based on Fluidized-Bed Extraction with Gas Chromatographic–Mass Spectrometric Detection

J. Am. Soc. Brew. Chem. 73(2):151-158, 2015 Linalool, an important substance in hoppy, aromatic beer, is one of the most aromatic components of essential oils in hops. Linalool concentration, among those of other substances, could be used to distinguish among hops varieties. As such, an effective, repeatable, and high-throughput method is required; a new method based on fluidized-bed extraction combined with detection by gas chromatography–mass spectrometry was developed and is presented in this study. This method is faster than the reference method, which uses steam distillation; it also reduces the possibility of thermal changes. Because this method saves organic solvent, energy, and sample amount, it can be considered a green method. Good repeatability of the method (8.0 mg/kg) was achieved with 3-hepten-1-ol as an internal standard. The detection limit of linalool is 1.0 mg/kg, and the limit of quantification is 3.5 mg/kg. Good agreement was achieved between the results of the new and steam-distillation methods. In addition, 59 samples of hops from four important Czech varieties (Agnus, Premiant, Saaz, and Sládek) from the 2013 harvest were analyzed, and the content of linalool was correlated with the content of bitter acids in xyz 3-D projection; four separate and clearly limited clusters that corresponded to the four tested varieties were obtained. Hops, along with malt and water, are the basic raw materials used for beer production. The basic role of hops is to provide beer with a pleasantly bitter taste and a hoppy aroma. This result is achieved within the wort boiling, during which poorly soluble α-acids are isomerized into the more-soluble iso-α-acids. The content of bitter substances (mostly α-bitter acids), therefore, is one of the key indicators of the quality of hops used for beer production (6). In addition to the bittering substances, hops contain other compounds that might affect the taste and flavor of beer. In particular, polyphenolic substances can modify the character of bitterness (25). Low-molecular-weight polyphenols can affect the sensory stability of beer indirectly due to their reducing capacity (8,26). Other very important ingredients are essential hop oils (0.5 to 3% w/w in hops), which are responsible for the smell of hops; the hop aroma is an important qualitative parameter that has a role in the selection of raw materials for brewing. The profile of hop essential oils is characteristic for each variety and can be used for variety identification (31). According to the chemical composition, compounds of essential oils can be divided into three basic groups: the hydrocarbon fraction, the oxygen fraction, and the fraction of sulfur compounds (5). Myrcene, α-pinene, farnesene, and humulene are the most important substances of the hydrocarbon fraction (70–80% essential oils) (5). The sulfur fraction contains only trace amounts of individual substances; however, due to their low flavor thresholds, they might significantly affect the taste and smell of beer, especially in a negative way. The oxygen fraction of essential oils is a mixture of alcohols, aldehydes, ketones, epoxides, esters, and acids. Due to their higher solubility in aqueous solutions, these substances might influence the flavor of beer in a significant way. Linalool is one of the major components of this group (5). Linalool is one of the most aromatic flavor components of hop essential oils, and, along with myrcene, it is considered the most odor-active volatile in all analyzed hop varieties (11,34,35). Thus, linalool is a primary substance in hoppy aromatic beers (11,19). It is a very flavorful terpene alcohol, with a citrusand bergamotlike odor. According to the literature (19,28), linalool is contained in hop essential oil in amounts up to 1.1% rel. The atlas of Czech hop varieties 2012 (7) states that this value in the current major Czech varieties (traditional Saaz and hybrid varieties Agnus, Premiant, and Sládek) is up to 0.7% rel. In addition to the flavor of hops, linalool is a significant part of several other aromas, such as fruit, spices, and chocolate, and it is widely used in the cosmetics industry. Its antiinflammatory properties also have a positive effect on human health (30). Although the concentration of linalool in beer is low, it can contribute to the overall flavor of the beer due to its synergistic effect with other hop essential oils. Higher content levels of linalool can be achieved by adding hops in the last part of hopping or by using dry hopping (13,16). The content of linalool, as well as the contents of other components, varies during the fermentation process; linalool is formed from geraniol and nerol and is simultaneously transformed into α-terpineol. This process is shown in Figure 1 (20,36). Linalool can also affect the sensation of off-flavors in finished beer. For example, Hanke et al (15) found that linalool, at a concentration near its threshold (~25 μg/L), suppresses the perception of dimethyl sulfide (DMS) in beer. However, higher concentrations of linalool (~70 μg/L) highlight the perception of DMS. A similar effect has been reported for isovaleric acid and diacetyl. Moreover, Hanke et al (14) found that hop essential oils, such as linalool, decrease the gushing tendency of beer, especially when hops are added in the late stages of wort boiling. Knowledge of the composition of hop secondary metabolites (essential oils, bitter acids, and polyphenols) is important not only for a qualitative description of the cultivar, but also because the spectrum of secondary metabolites can be used to differentiate among hop varieties. Several profiling methods of authenticating hop varieties using the polyphenol spectrum have been published (24,29). Linalool, along with a wide spectrum of hop compounds, can be used to identify the hop cultivar. In 1998, 1 Corresponding author. E-mail: [email protected]; phone: +420 224 900 150. http://dx.doi.org/10.1094/ASBCJ-2015-0406-01 © 2015 American Society of Brewing Chemists, Inc. 152 / Štěrba, K., Čejka, P., Čulík, J., Jurková, M., Krofta, K., Pavlovič, M., Mikyška, A., and Olšovská, J. Perpete et al (31) endeavored to differentiate hop pellets by essential oil analysis. Volatile compounds of five aroma cultivars (Styrie, Saaz, Lublin, Mount Hood, and Hallertau) and seven bitter cultivars (Northern Brewer, Nugget, Pride of Ringwood, Northdown, Galena, Target, and Challenger) were extracted with a Likens-Nickerson simultaneous solvent extractor, separated by gas chromatography (GC), and identified by mass spectrometry (MS). High levels of bergamotene and farnesene were found only in Saaz, Lublin, and Styrie samples. Quantification of 4-decenoic acid methyl ester and 3-methyl butyl isobutyrate proved to be a quick means of distinguishing between non-European and European bitter hops from aroma cultivars. Kovačevič et al (22) conducted statistical processing of the resulting fingerprints using the classical method, steam distillation with GC-MS, for the analysis. Based on the fact that the composition of essential oil in hops depends more on hop variety and less on growing, processing, and storage conditions, the fingerprints of 16 representative components of essential oils were correlated with a sample variety. Seventy-eight samples belonging to the five most important hop varieties (Aurora, Bobek, Celeia, Magnum, and Savinjski) grown in Slovenia were analyzed. The methods used, cluster analysis and principal component analysis, reliably differentiated the five varieties. Jelínek et al (17) compared the composition of the secondary metabolites αand β-bitter acid analogues, essential oils, and polyphenols to perform a varietal differentiation of seven Czech hop varieties (dry Agnus, Bor, Harmonie, Premiant, Rubín, Sládek, and Saaz hop cones). They concluded that hop essential oil constituents contribute significantly to the individual hop varieties. They also established a dichotomous key for the authentication of Czech hop varieties based on some characteristic varietal markers. The authors used classical analysis with a steam distillation step in the sample preparation. Several methods of determining linalool and other essential oils have been published; the methods differ mainly in the preparation step, which affects the recovery of the method. The last step of the method is GC with MS or flame ionization detection (FID). The reference methods, ASBC/Hops-17 and EBC 7.10 and 7.12 (3,9) are based on the steam distillation of essential oils from a mixture of ground hop material (more than 100 g of the sample) and a large volume of water. The oil floating on top of the mainly aqueous condensate is analyzed by GC-FID. The disadvantages of this method are the time-consuming nature of the procedure, which entails 3 to 4 h of distillation, and the tendency of essential oils (including linalool) to undergo changes in chemical composition during this long process (2,32). One way to reduce the effects of oxygen and high temperatures on the chemical composition during extraction is through the use of supercritical hops extraction. In the present study, the sample was extracted by CO2 (4) and subsequently extracted on a solid phase extraction (SPE) column (12). The disadvantages of this procedure are the limited extraction ability of polar compounds and an instrumental challenge (1). Several authors have described methods based on a new principle, headspace solid-phase microextraction (SPME), which is a relatively simple procedure. The hops sample is milled and inserted into the headspace vial, and GC analysis is immediately performed (10,21). The disadvantage of this method is that the amount of analyte absorbed on a fiber depends on the extraction conditions, age of the fiber, and matrix effect (33). Another possible method of extracting the essential oils is the use of ultrasound. Aberl et al extracted oils from hops using ethanol during sonication of the sample, and the resultant extracts were analyzed by headspace GC-MS (2). As mentioned previously, identification of the hop cultivar and the description of its qualitative parameters are very topical and important questions, both for hop producers, who need this information for distinguishing purposes and for qualitative descriptions of the cultivars, and for brewers, who must choose quality raw materials to maintain consistent beer quality. Therefore, the development of a high-throughput, reproducible, and effective method for this purpose is desired. Thus, the aim of this study was to develop a method of determining linalool in hops using fluidized-bed extraction. This method was chosen because the principle behind it meets the requirements of an effective and high-throughput method, with its low sample and solvent consumption levels and short heating time of the sample at low temperatures, which leads to fewer thermal changes in the sample composition. The correlation between the new method and reference methods based on steam distillation was proven using eight samples (varieties Saaz, Sládek, Premiant, and Agnus, each from two different localities). In addition, the content levels of linalool in 59 hops samples from four important Czech varieties from the 2013 harvest were analyzed and correlated with the content levels of bitter acids. The probability of using linalool as a marker of hop variety was suggested and is discussed.