The detectives determine vicinal diketones (VDK) in beer
Chapter 35
📂 Case overview: More than a year since the opening of their agency, the food detectives come face to face with an arch nemesis, Micobiarties, microbial contamination! A client complains his beer is frequentgly affected by microbial contamination during the production process. Miss Mapple comes to the rescue by suggesting using vicinal diketones as quality indicators. The team goes one step further and pits steam distillation & colorimetric analysis against gas chromatography for VDK determination. Which method wins the fight against Microbiarties? Read on to find out.
Shallot Holmes enters the office with a gloomy face. He gathers his team around and announces that they have a very serious case that bears all the markings of an arch-nemesis in the making. A customer has been having frequent cases of microbial contamination in his brewery. But how can the team defend against these Microbiarties?
Miss Mapple perks up as soon as she hears the case is about beer. After all, she is known to enjoy a good glass of cider. And she was instrumental in a case of using the Kjeldahl method for protein determination in beer. The team fully agrees she should lead this case.
Miss Mapple takes a few hours to prepare. Then she calls in the team for her briefing and ideas on how to help the client.
She gets right into it by suggesting one approach to detecting microbial contamination: determination of vicinal diketones (VDK). Because she notices a few of her colleagues scratching their heads, she gives a quick explanation of vicinal diketones.
In beer fermentation, two vicinal diketones, diacetyl (2,3-butanedione) and 2,3-pentanedione, are formed. VDK influence beer flavor, where concentrations above their flavor threshold (approx. 0.1 mg/L for diacetyl and 1.0 mg/L for 2,3-pentanedione) lead to undesired butter/butterscotch-like flavors. The concentration of VDK depends on several process conditions, including:
- Wort composition
- Fermentation regime
- Yeast strain
- Microbial contamination
It is the last point that is most relevant to their case and fighting nasty Microbiarties. Microbial contamination, or no microbial contamination, control of VDK levels during fermentation is a very important aspect of quality control in a brewery.
Methods for determination of vicinal diketones
Levels of vicinal diketones can be determined by a variety of analytical methods. Two approaches are most frequently used and come recommended by the European Brewery Convention (EBC):
- Colorimetric assays, such as complex formation with o-phenylenediamine – where the VDK concentration is reported as a sum of both volatiles and allows no differentiation of diacetyl and 2,3-pentanedione
- Gas chromatography (GC) – where diacetyl and 2,3-pentanedione can be separated and quantified individually
Miss Mapple points out that typically the concentration of diacetyl is much greater than the concentration of 2,3-pentanedione and diacetyl exerts a greater influence on flavor. Because of this, the colorimetric assay is widely used in the brewing industry.
She suggests that the team tries out both recommended methods. Firstly, they want to distill VDK standards and beer samples, then determine the content of vicinal diketones, in complex with resp. the complex with o-phenylenediamineis, with a UV-Vis spectrophotometer. Then they want to compare their results to the other accepted method for determination of vicinal diketones, GC analysis.
They get to work immediately. There is no time to lose in their fight against Microbiarties.
For the colorimetric analysis, they use a steam distillation system to prepare their samples, and a spectrophotometer to read the absorbance values of their standards and beer samples. They base their method on a distillation application note Miss Mapple has found. The detectives make sure to distill VDK standards at a level of 0.1 mg/L of each diacetyl and total 2,3-pentanedione with different percentage of steam power settings to validate the efficiency of their distillation. They also make use of a few cue cards to optimize their distillation method.
For their sample, the detectives select a Czech Pilsner beer, as these types of beer are typically characterized by elevated VDK levels. The detectives analyze the sample using different % steam power settings on their system and by using gas chromatography (GC). They perform the GC determination of diacetyl and 2,3-pentanedione in accordance with EBC 9.24.2.
Hurriedly, they look at their final data. Firstly, they examine how the two methods compare for vicinal diketone standards, consisting of aqueous solution of 0.1 mg/L diacetyl and 0.1 mg/L 2,3-pentadione.
| Determination | Steam Distillation: % Steam Power | Steam Distillation: VDK (mg/L) | Steam Distillation: % Recovery | Gas chromatography: Diacetyl (mg/L) | Gas chromatography: 2,3-pentadione (mg/L) | Gas chromatography: VDK (mg/L) |
|---|---|---|---|---|---|---|
| 1 | 10 | 0.201 | 100.3 | 0.110 | 0.100 | 0.210 |
| 2 | 10 | 0.201 | 100.3 | 0.110 | 0.100 | 0.210 |
| 3 | 10 | 0.201 | 100.3 | 0.110 | 0.100 | 0.210 |
| 1 | 50 | .199 | 99.1 | 0.110 | 0.100 | 0.210 |
| 2 | 50 | .199 | 99.1 | 0.110 | 0.100 | 0.210 |
| 3 | 50 | .196 | 99.1 | 0.110 | 0.100 | 0.210 |
| 1 | 100 | .199 | 99.1 | 0.110 | 0.100 | 0.210 |
| 2 | 100 | .197 | 99.1 | 0.110 | 0.100 | 0.210 |
| 3 | 100 | .198 | 99.1 | 0.110 | 0.100 | 0.210 |
Miss Mapple happily tells the team that when conducting distillation of vicinal diketones with a classical Parnas or Markham still, a heating period of 6 minutes and a distillation time of 8 to 10 minutes is needed to recover the VDK. Using their distillation unit has enabled the detectives to use steam power varying from 10 to 100% and achieve complete recovery of VDK in approximately five 1 min. distillation times.
The food detectives turn their attention to the Czech Pilsner beer sample:
| Determination | Steam Distillation: % Steam Power | Steam Distillation: VDK (mg/L) | Gas chromatography: Diacetyl (mg/L) | Gas chromatography: 2,3-pentadione (mg/L) | Gas chromatography: VDK (mg/L) |
|---|---|---|---|---|---|
| 1 | 10 | 0.090 | 0.041 | 0.034 | 0.075 |
| 2 | 10 | 0.089 | 0.041 | 0.034 | 0.075 |
| 3 | 10 | 0.089 | 0.041 | 0.034 | 0.075 |
| 1 | 50 | .095 | 0.040 | 0.032 | 0.072 |
| 2 | 50 | .093 | 0.040 | 0.032 | 0.072 |
| 3 | 50 | .084 | 0.040 | 0.032 | 0.072 |
| 1 | 100 | .088 | 0.036 | 0.032 | 0.068 |
| 2 | 100 | .085 | 0.036 | 0.032 | 0.068 |
| 3 | 100 | .088 | 0.036 | 0.032 | 0.068 |
There, they note that the % steam power setting has no significant effect on the vicinal diketone determination and the results are similar to those of their GC analysis. In fact, the differences in the results between steam distillation and GC fall into the range or reproducibility R95. R95 refers to the allowed tolerance when two different laboratories carry out the analysis on the same sample with the exact same method. The team here instead present results for the same sample using two different methods and still obtain results under the allowed tolerance.
The food detectives summarize their findings and discuss which method to recommend to their customer. Miss Mapple concludes the following:
- In comparison to classical Parnas or Markham stills, the distillation with a modern steam distillation system can be executed faster and more flexibly in terms of steam power
- Compared to GC analysis, steam distillation is faster, easier and does not need trained personnel to carry out the analysis
The team then decides to recommend steam distillation to their client for keeping Microbiarties in check and for controlling beer flavour. The client is pleased with their suggestions and implements the method. Microbiarties has been beaten in this brewery, but will this be the last the food detectives hear of the villainy of microbial contamination? Unlikely…but armed with knowledge and methods, they are ready for the next battle!