Simultaneous Online Monitoring and Sterility Assurance in Aseptic Food Processing Based on a Combined Calorimetric Gas- and Spore-Based Biosensor Array

Abstract

Introduction Gaseous hydrogen peroxide is one of the most used sterilants for packaging in aseptic filling machines [1]. To validate a sterilization method under investigation, spores (resistant microorganisms against the sterilant) are exposed to the sterilant, the logarithmic kill rate is determined and at the end, the sterilization can be concluded. The main drawback of these methods is their tedious physical work and their slow response; the results can be obtained at the earliest after 24-48 hours. As a result, a novel sensing method has been previously introduced [2] to overcome these issues. In this work, such spore-based biosensor is combined with a calorimetric H2O2 sensor onto single chips to evaluate both the viability of spores and to determine gaseous hydrogen peroxide concentrations. Materials and Methods The calorimetric gas sensor was developed to measure gaseous hydrogen peroxide concentrations. Here, two meander structures were utilized as temperature sensors (see Fig. 1 a), at the bottom part of the sensor chip). One sensor was used as a reference (i.e., inert to H2O2 exposure), whereas the other one was employed as an active element (i.e., functionalized with MnO2 which catalyzes H2O2). As both structures were exposed to H2O2, a temperature increase occurred only at the active sensor. As a result, a temperature difference between both sensors could be correlated to the H2O2concentration. In addition, a spore-based biosensor was fabricated to evaluate the viability of spores. For this, interdigitated electrodes were operated as impedimetric sensors (see Fig. 1 a) at the top part of the sensor chip). A differential setup measurement was used similar like in the calorimetric gas sensor. In this case, spores were immobilized on the active part of the sensor chip. Upon H2O2 exposure, changes in the morphology of the spores aroused, which (by means of impedimetric changes) could be correlated to the viability of spores and hydrogen peroxide concentration. Results and Conclusions The calorimetric gas sensor responds to different concentrations of gaseous hydrogen peroxide (see Fig. 1 b)). The upper curves correspond to the temperature of the catallytically active sensor and the passive one. The temperature difference and accordingly, the actual H2O2 concentration is shown in the lower part curve. In the second experiment, biosensors were investigated with three different strains of bacteria measured at different stages. In the first stage, three biosensors’ impedances were measured after being cleaned. In the second step, B. atrophaeus DSM 675, B. subtilisDSM 402 and G. stearothermophilus DSM 5934 spores were immobilized on them. Finally, the spore-based biosensors were submitted to several concentrations of gaseous hydrogen peroxide. Signal responses (impedance) of the spore-based biosensors at different applied H2O2 concentrations are overviewed in Fig. 1 c).In conclusion, the synergy of these two sensor types as one combined sensor array allows a considerably more specific multi-parameter experience in aseptic filling machines in comparison to isolated microbiological state-of-the-art- or hydrogen peroxide methods.Fig. 1. a) Combined sensor array consisting of a calorimetric H2O2 sensor and a spore-based biosensor with their respective sensor calorimetric and impedance responses in b) and c).