GERSTEL Solutions No. 7 (pdf; 1,86 MB)

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GERSTEL Solutions Worldwide Application Foto: wikipedia Extraction technique comparison Beer flavor analysis with a twist Hops contain terpenoids that help give beer its slightly bitter taste and, if properly combined and controlled, a fine flavor. Since variations in terpenoid concentrations occur during the beer brewing process, careful monitoring and adjustment is required in order to achieve consistent quality. The Asahi Breweries has succeeded in determining the concentrations of a long list of relevant flavor compounds in just one analysis. To achieve this, Asahi uses Stir Bar Sorptive Extraction (SBSE). Beer is one of the oldest and most widely enjoyed beverages in the world. Traditionally, beer is based on four ingredients: Water, barley, yeast and hops. The barley is allowed to germinate and is then malted, that is, dried at elevated temperatures; water is added, and the malt boiled to form the wort. Hops are added as the wort boils. After filtering, yeast is added and the fermentation process puts its finishing touches to the product. The temperature used during the malting process as well as the hops, or rather the terpenoids they contain, are some of the key factors in forming the distinct taste of the brew. Hops and malt additionally serve as natural preservatives. During the various stages of the brewing process, variations in terpenoid concentrations can occur. Careful monitoring and adjustment is required in order to achieve consistent quality. The Asahi Breweries are able to determine the concentrations of a long list of terpenoids in modestly sized samples of beers at all stages of production. This requires a sensitive technique, especially when analyzing beer with mild hop flavor, which is typically preferred in Japan. Asahi relies on GC/MS in combination with Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister®. Mr. Toru Kishimoto and his colleagues from the Asahi Research and Development Centre have reported their findings regarding the advantages of the Twister technology in comparison with other techniques, in the Journal of Agricultural and Food Chemistry [2005; 53(12); 4701-4707]. Conventional techniques are not always up to the task While there is general agreement that hop flavor compounds should be determined using GC/MS, opinions vary when it comes to choosing the best extraction technique. Different laboratories have chosen a wide variety of techniques to extract and concentrate hop flavor compounds from beer in its various stages of production: Lam et al. [1] let two liters of beer run through a silica gel column to separate and determine linaloole, geraniole and - citronellole that add citrus and floral flavor notes. Steinhaus and Schieberle [2] extracted flavor compounds directly from hops and identified linaloole and myrcene as potential flavour compounds. De Keukeleire et al. [3] extracted a variety of hops using Supercritical Fluid Extraction, based on CO 2 , enabling them to identify myrcene, -Caryophyllene, -humulene and -farnesene. In addition to the above, Irwin [4] and Goiris et al. [5] used a silica gel column to separate flavor compounds found in Hersbrucker Beer; demonstrating clearly that the oxygenated sesquiterpene fraction contributes to the “hoppy” character of the beer. This group includes eudesmole, humulene epoxide and humulenole II among others. Lermusieau et al. [6] used an XAD 2 resin column to determine that linaloole and -Damascenone are active flavor compounds. Though all the above approaches provide meaningful results, they still leave a lot to be desired according to Toru Kishimoto and his colleagues.: „These techniques require raw hops and large volumes of sample in order to extract the active flavor compounds in sufficient amounts. Furthermore, these techniques work mainly for beers that have a strong hop flavor”. 4 GERSTEL Solutions Worldwide – May 2007
GERSTEL Solutions Worldwide Application In order to accurately determine the terpenoid profile of Japanese beers, that typically have less intense hop flavor, a more sensitive analysis technique is needed. According to the Asahi scientists, conventional extraction techniques have proven quite complex and labor intensive and are therefore not well suited for routine analysis involving large numbers of samples. One promising alternative is already used by Asahi to establish terpenoid profiles: Stir Bar Sorptive Extraction (SBSE). This technique has previously proven its worth for the determination of trace concentrations of VOCs in beer [7,8]. In practice, SBSE is performed using the GERSTEL Twister, a magnetic stir bar with a layer of between 24 and 126 μL PDMS sorbent depending on the type. “The technique is really easy to handle, up to 1000 times more sensitive than SPME and readily automated”, Toru Kishimoto and his colleagues stated. SBSE compared with Conventional extraction In order to determine whether SBSE was up to the task, the Asahi scientists compared results from SBSE and a widely used methylene chloride based liquid/liquid extraction. Samples used for the comparison were two commercially available Japanese beers, one with intense hop flavor, and one with mild hop flavor. The analysis was performed using GC/MS; compounds identified were quantified using the response factor for -damascenone, added as an internal standard. Results: Using conventional methylene chloride extraction, only three compounds SBSE is performed using the patented Twister ® . The Twister is a magnetic stir bar, coated with glass and surrounded by a PDMS phase of up to 126 μL. The Twister is immersed into an aqueous or other liquid sample, in this case into the beer or wort sample, from which it efficiently extracts especially the hydrophobic analytes while stirring. The efficiency of SBSE for a given compound correlates well with the partition coefficient for that compound between polydimethylsiloxane (PDMS) and water, which in turn correlates well with its Octanol/Water partition coefficient K O/W . K O/W is a physical parameter used to describe the hydrophilic or hydrophobic properties of a chemical compound including its tendency to accumulate in fat tissue [10]. The logarithm of the PDMS/water partition coefficient is almost equal to the logarithm of the octanol/water partition coefficient (Log K O/W ), that can be found in standard tables. A large Log K O/W value signifies a high Background on Stir Bar Sorptive Extraction degree of hydrophobicity, indicating that the analyte is highly soluble in PDMS and can be extracted with good recovery by the Twister ® . „The PDMS/water phase system reaches equilibrium faster at higher temperatures“, Toru Kishimoto and his colleagues report in the Journal of Agricultural and Food Chemistry. They chose a temperature of 40 °C, extracting the sample over two hours; the Log K O/W values of the terpenoids ranged from 3.4 to 7.1 and -damascenone was used as internal standard since its Log K O/W value of 4.4 was well within this range. Further, a strong correlation was observed between the response factors of -damascenone and all other terpenoids. The Twisters were subsequently automatically desorbed using an MPS fitted with Twister Option in combination with a Thermal Desorption Unit (TDU). GC/MS was performed using a GC 6890/MSD 5973N system from Agilent Technologies. were identified: Linaloole, geraniole und - eudesmole. The limited extraction efficiency was attributed to interference from matrix components such as proteins, amino acids and polyphenols. On the other hand, using SBSE, Kishimoto and his colleagues were able to identify a large number of other terpenoids, among them -eudesmole, humulene, humuleneepoxide I, -farnesene, caryophyllene and geraniole. “The results were clear: The SBSE method enables us to easily determine a number of compounds that we were not able to find using the conventional extraction method.” Tracking terpenoid levels throughout the brewing process The simplicity of the SBSE method and the fact that it requires only a small sample size and very little sample preparation, allowed the Asahi scientists to monitor both the presence and the changes in concentrations of terpenoids during the wort boiling process. Hops normally contain high levels of caryophyllene, humulene and - Farnesene; however, during the wort boiling process, only small concentrations are Toru Kishimoto, Laboratory Manager at Asahi R&D Centre, discusses results from hop flavor analysis performed using the GERSTEL Twister with a co-worker. GERSTEL Solutions Worldwide – May 2007 5
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