Resumo do Pôster:
1. Introduction
Bitterness from beer is traditionally originated from iso-alpha acids formed from boiling of hops. Pre-isomerized hop products have been developed to utilize hops more cost efficiently and to improve the brewing processes. Typically, e.g. iso-pellets are added to the wort or e.g. Iso, Rho, Tetra or Hexa products are added after fermentation as downstream products, meaning as post-fermentation bittering products. These hydrogenated, also called reduced iso-alpha acids (Tetra, Rho and Hexa), are used to achieve light stability of beer. Hexa and Tetra are also known to be relative non-polar and therefore effective as a foam stabilizer. It is recommended that Tetra and Hexa should not be used in higher concentrations than approximately 7 mg/L, as above this, the beer foam becomes unnatural1.
Beer bitterness is typically routinely analyzed with two techniques: Manual/automated isooctane extration or with HPLC. In this work the bitterness analysis is fully automated with an automated discrete photometric analyzer. Acidified beer/wort is run through a solid phase extraction column that binds bittering compounds. Bound material is eluted and eluent is measured at 275 nm.
2. Purpose
To study the recovery of the reduced iso-alpha acids Tetra, Hexa and Rho with a novel approach for bitterness analysis based on a fully automated solid phase extraction and photometric measurement method.
3. Method
Manual iso-octane method was modified from the official EBC method2: 2 mL sample, 0.1 mL 6 M HCl and 4 mL iso-octane were pipetted in a 20 mL Erlenmayer flask. The flask was put on a horizontal shaker for 30 minutes at 250 rpm. After shaking the iso-octane was poured directly from the flask to the measuring cuvette.
In the automated method a discrete photometric analyser fully automate the bitterness analysis. A degassed sample is inserted into the analyzer where bittering substances are extracted using SPE column. The acidified sample is aspired to the column where the bittering substances are bound to the column. The sample matrix is then washed out of the column. Next, the analytes are released from the column by aspirating eluent, which is automatically pipetted to the measuring cuvette within the analyzer and absorbance at 275 nm is read. After elution, the column is flushed twice with eluent to clean the column. In the Tetra/Hexa measurement the sample volume was 600 μL. In the Iso/Rho measurement the sample volume was 150 μL. The different sample volumes were used because the Tetra/Hexa concentrations were very low whereas in the Iso and Rho measurement high sample concentrations were analyzed. For the measurements three replicates were made of each sample.
4. Results and Discussion
Tetra and Hexa response compared to Iso-alpha acid: water based standard samples containing 2-10 mg/L Tetra- or Hexa-iso-alpha-acids were analyzed with both the manual iso-octane method and the automated method using a 600 μL sample volume. The recovery of Tetra, Hexa and Iso was slightly different in the EBC manual isooctane method compared to the automated bitterness determination. If the automated method is calibrated with an Iso containing sample or Iso containing standard solution, and a sample containing Tetra is analyzed, the result will be approximately 20 % higher in the automated method compared to the manual Iso-octane extraction. If the automated method is calibrated with Tetra containing sample or standard solution there is no difference to the manual method. Likewise, if the automated method is calibrated with an Iso containing sample or Iso containing standard solution and a sample containing Hexa is analyzed, the result will be approximately 10% lower in the automated method compared to the manual iso-octane extraction. If the automated method is calibrated with Hexa containing sample or standard solution there is no difference to the manual method.
Rho response compared to Iso-alpha acid: water based standard samples containing 10-60 mg/L Rho-iso-alpha-acids were analyzed with both the EBC manual iso-octane method and the automated method using a 150 μL sample volume. The recovery of Rho differed significantly from the recovery of Iso in the automated bitterness determination compared to the manual EBC iso-octane method. If the automated method is calibrated with an Iso containing sample or Iso containing standard solution, and a sample containing Rho is analyzed, the result in the automated determination will be approximately 60% of the result from the manual EBC iso-octane extraction. If the automated method is calibrated with Rho containing sample or standard solution there is no difference to the manual method.
The tasted bitterness per mg/L was compared to the relative responses of the iso-octane and the automated methods per mg/L. The differences in relative responses in iso-octane and automated methods were caused by the differences in molar absorptivities at 275 nm or incomplete extraction efficiencies. However, the automated relative responses of Rho and Tetra was closer to the relative taste-intensities of Rho and Tetra than the iso-octane method.
5. Conclusion
The recoveries of different types of iso-alpha acids are different in automated compared to the manual EBC iso-octane extraction. Therefore, whenever possible the automated method should be calibrated with samples or standard solutions containing similar type of iso-alpha acid as is in the sample to be analyzed. It is possible also to use a single calibration for all types of iso-alpha acids, but then different recoveries comparing to the isooctane extraction will be seen. If the method is calibrated with Iso, Tetra will give approximately 20% higher results, Hexa will give approx 10% lower results and Rho will give approx 40% lower results when compared to the iso-octane method.
In the analyzer multiple methods with different calibrations are easy to set-up. Flexible software allows also a calibrator factor to be used. If the analyzer analytical results are compared to the sensory tests, these water based studies shows promising correlation of the results. In the future, these findings need to be verified with real beer samples to see how the beer matrix effects to the results. The experiments in this paper were made with water based standard solutions. Further experiments are needed to study the extraction of non-iso-alpha acid bittering compounds present in real beer and wort samples and to see the effect of other matrix components to the extraction efficiencies.
6. References
- Biendl, Martin. Pre-isomerized hop products –potential and practical use. Brauwelt International, 2002/IV, 20-25.
- Iso-octane extraction - Bitterness from beer and wort. EBC (8.8, 9.8), MEBAK (2.18), ASBC (Beer-23).
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