Acetoin Biosensor Based on Acetoin Reductase-Modified Field-Effect Sensor Applied for Acetoin Detection in Beer Samples
Authors: Jablonski Melanie , Muenstermann Felix , Molinnus Denise , Muschallik Lukas , Bongaerts Johannes , Wagner Torsten , Keusgen Michael , Siegert Petra , Schoening Michael J. 
Published: May 2020

Introduction

In food industry, the use and dosing of flavoring substances is an important aspect during fabrication processes. Acetoin and diacetyl are flavoring agents which can be found in many food products, like yogurt, cheese, milk, wine and beer [1]. In beer, acetoin/diacetyl is a critical parameter for the maturation degree of the beverage during fabrication. They are formed during the first phase of beer fermentation and getting reduced during the later phase of fermentation and the maturation. Both can have a negative effect on the beer flavor, when they are above a certain concentration. Typical acetoin concentrations in beer are in the range of 10-50 µM. Measuring the acetoin/diacetyl concentration during the production can serve as a quality control and unnecessary maturation time could be avoided [2]. Until now, the quantitative determination of the acetoin/diacetyl concentration is mostly done “off-line” by gas chromatography. Therefore, a sample has to be sent to an external laboratory, which takes time till results are achieved, or the brewery needs an own laboratory, whereby enormous costs arise and trained staff is needed. Because of that, we recently developed a biosensor for the detection of acetoin based on an enzyme-modified electrolyte-insulator-semiconductor (EIS) sensor, which was characterized in buffer solutions [3]. In this work, the novel biosensor was applied for acetoin measurements in beer samples for the first time.

Materials and Methods

As sensor structures, capacitive Al-p-Si-SiO2-Ta2O5 field-effect sensors have been used, schematically shown in Fig. 1(a). The Ta2O5 transducer surface was modified with a novel acetoin reductase by means of cross-linking. The acetoin reductase (from B. clausii DSM 8716T) catalyzes the reduction of acetoin to 2,3-butanediol, while NADH serves as a cofactor [4]. The fabricated acetoin biosensors have been electrochemically characterized by capacitance-voltage- and constant-capacitance (ConCap) measurements in buffer solution (0.2 mM Tris-HCl) with varying acetoin concentration and beer samples, spiked with different amounts of acetoin.

Results and Conclusions

The acetoin sensitivity was investigated in beer solutions and the biosensors showed a linear behavior in the concentration range between 10 µM and 90 μM with a sensitivity of ca. 30 mV/dec, see Fig. 1(b). A detailed description of experiments and the obtained results will be presented and discussed.
Figure 1: Schematic measurement set-up with the layer structure of the EIS sensor modified with acetoin reductase (a). Exemplary ConCap-measurement curve recorded in buffer-beer mixture (ratio 20:1) spiked with different acetoin concentrations, respectively (b).

Acknowledgments

The authors thank the ministry of innovation, science and research of the state of North-Rhine-Westphalia for financial support (funding program FH-Struktur 2016).

References

[1] P. Li, Y. Zhu, H. Shun, J. Fan, Q. Hu, Y. Cao, Development and validation of high-performance liquid chromatography method for the determination of diacetyl in beer using 4-nitro-o-phenylenediamine as the derivatization reagent, Journal of Agricultural and Food Chemistry 60 (2012) 3013–3019. doi: 10.1021/jf3007163.
[2] A. D. Haukeli, S. Lie, Formation and removal of acetoin during yeast fermentation, Journal of the Institute of Brewing 81 (1975) 58–64. doi: 10.1002/j.2050-0416.1975.tb03662.x.
[3] D. Molinnus, M. Muschallik, L. O. Gonzalez, J. Bongaerts, T. Wagner, T. Selmer, P. Siegert, M. Keusgen, M. J. Schöning, Development and characterization of a field-effect biosensor for the detection of acetoin, Biosensors and Bioelectronics 115 (2018) 1–6. doi: 10.1016/j.bios.2018.05.023.
[4] L. Muschallik, D. Molinnus, J. Bongaerts, M. Pohl, T. Wagner, M. J. Schöning, P. Siegert, T. Selmer, (R,R)-Butane-2,3-diol dehydrogenase from Bacillus clausii DSM 8716T: Cloning and expression of the bdhA-gene, and initial characterization of enzyme, Journal of Biotechnology 258 (2017) 41– 50. doi: 10.1016/j.jbiotec.2017.07.020.

Figure 1